Targeted Inhibition Using Engineered Oligonucleotides

ABSTRACT

Disclosed herein are engineered oligonucleotides for selective inhibition of polypeptide expression and activity. Also disclosed herein are methods of selectively inhibiting polypeptide expression and activity contacting an engineered oligonucleotide with a polynucleotide encoding the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS(S)

This application is a Continuation Application of U.S. application Ser.No. 17/692,644, filed Mar. 11, 2022, which is a Continuation Applicationof International Application No. PCT/US2021/025639, filed on Apr. 2,2021, which claims the benefit U.S. Provisional Application No.63/004,045, filed Apr. 2, 2020, the disclosures of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.U43CA221567 awarded by the National Cancer Institute and support fromSBIR Contract No. HHSN272201800034C awarded by the National Institute ofAllergy and Infection Disease. The Government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in xml format and is hereby incorporated byreference in its entirety. Said xml copy, created on Jan. 17, 2023, isnamed 199259_701303_SL.xml and is 4,372,991 bytes in size.

SUMMARY

Disclosed herein are engineered oligonucleotides or salts thereof thatcan comprise a polynucleotide sequence. In some embodiments, anengineered oligonucleotide or salt thereof can be at least partiallycomplementary to at least a portion of at least a first and a second RNAoriginating from two genetic loci that are associated with a disease orcondition. In some embodiments, when an engineered oligonucleotide orsalt thereof at least partially binds to a first RNA: a first region ofat least seven contiguous bases in the engineered oligonucleotide can becomplementary to contiguous nucleic acids contained within the first RNAand a second region of at least five contiguous bases in the engineeredoligonucleotide can be complementary to contiguous nucleic acidscontained within the first RNA. In some embodiments, when an engineeredoligonucleotide or salt thereof at least partially binds to a secondRNA: a first region of at least seven contiguous bases in the engineeredoligonucleotide can be complementary to contiguous nucleotides containedwithin a second RNA, and a second region of at least five contiguousbases in the engineered oligonucleotide can be complementary tocontiguous nucleotides contained within the second RNA. In someembodiments, a predicted Gibbs free energy (ΔG) of binding of anengineered oligonucleotide to a first and a second RNA can range,individually, from about −17 to about −36 kcal mol⁻¹ at about 37 degreesCelsius and at a pH ranging from about 7.2 to about 7.6. In someembodiments, an engineered oligonucleotide or salt thereof can be anantisense oligonucleotide, a synthetic microRNA (miRNA), or a smallinterfering RNA (siRNA). In some embodiments, an engineeredoligonucleotide or salt thereof can comprise one or more nucleotideinsertions, nucleotide deletions, nucleotide substitutions, or anycombination thereof, relative to one or more otherwise comparablenon-coding RNAs (ncRNAs). In some embodiments, an engineeredoligonucleotide or salt thereof, when at least partially bound to thefirst or the second RNA, can have an at least about 10% lower Gibbs freeenergy (ΔG) of binding at about 37 degrees Celsius and at a pH rangingfrom about 7.2 to about 7.6, relative to a ΔG of binding of theotherwise comparable ncRNA binding to the first or the second RNA atabout 37 degrees Celsius and at a pH ranging from about 7.2 to about7.6. In some embodiments, an engineered oligonucleotide or salt thereofcan be from about 5 to about 50 nucleotides in length. In someembodiments, an engineered oligonucleotide or salt thereof can comprisea ribose sugar. In some embodiments, an engineered oligonucleotide orsalt thereof can comprise a deoxyribose sugar. In some embodiments, anncRNA can be an miR-30 mircro RNA (miRNA), an miR-29 miRNA, an miR-26miRNA, an miR-27 miRNA, an miR-101 miRNA, an miR-145 miRNA, an miR-205miRNA, an miR-338 miRNA, or an miR-375 miRNA. In some embodiments, anengineered oligonucleotide or salt thereof can have at least 90%sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 245, SEQ ID NO:246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO:255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO:465, SEQ ID NO: 624, SEQ ID NO: 625, SEQ ID NO: 626, SEQ ID NO: 627, SEQID NO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO:632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO:641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 840, SEQ ID NO: 841, SEQID NO: 842, SEQ ID NO: 843, SEQ ID NO: 844, SEQ ID NO: 845, SEQ ID NO:901, SEQ ID NO: 902, SEQ ID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905, SEQID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO:910, SEQ ID NO: 911, SEQ ID NO: 912, SEQ ID NO: 913, SEQ ID NO: 914, SEQID NO: 915, SEQ ID NO: 916, SEQ ID NO: 917, SEQ ID NO: 918, SEQ ID NO:919, SEQ ID NO: 920, SEQ ID NO: 921, SEQ ID NO: 922, SEQ ID NO: 923, SEQID NO: 924, SEQ ID NO: 925, SEQ ID NO: 926, SEQ ID NO: 927, SEQ ID NO:928, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 932, SEQID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO:937, SEQ ID NO: 938, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQID NO: 942, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, SEQ ID NO:946, SEQ ID NO: 947, SEQ ID NO: 948, or SEQ ID NO: 949, as determined bya BLAST pairwise sequence alignment algorithm. In some embodiments, anengineered oligonucleotide can form a secondary structure comprising astem-loop in an aqueous solution at a temperature ranging from about 15degrees Celsius to about 37 Celsius at a pH ranging from about 6.5 toabout 7.6. In some embodiments, an engineered oligonucleotide or saltthereof can comprise a chemically modified base, chemically modifiedsugar, chemically modified backbone or phosphate linkage, or anycombination thereof relative to a naturally occurring base, sugar,backbone, or phosphate linkage. In some embodiments, a chemicalmodification can be selected from the group consisting of: a methylgroup, a fluoro group, a methoxyethyl group, an ethyl group, ahydroxymethyl group, a formyl group, bridged nucleic acid, lockednucleic acid, a carboxylic acid or salt thereof, a phosphothionatemodified backbone, a methylphosphonate modified backbone, an amino-alkylchain modification, and any combination thereof. In some embodiments, anengineered oligonucleotide or salt thereof, when chemically modified,can comprises the formula: (N)_(a)(mN)_(b)(N)_(c)NN;(N)_(a)(mN)_(b)(N)_(c)sfNsmN; or (fNmN)_(h)(mN)_(i)(fNmN)_(j)sfNsmN;wherein each N can be independently uracil, guanine, adenine, cytosine,or other natural nucleotide; each mN can be independently a2′-O-methyl-modified uracil, guanine, adenine, or cytosine; each s canbe independently a phosphothionate-modified backbone; each fN can beindependently 2′fluoro-modified uracil, guanine, adenine, or cytosine;and each a can be from 8-10, each b can be from 7-10, each c can be from2-4, each h can be from 5-7, each i can be 0 or 1, and each j can befrom 3-4. In some embodiments, an engineered oligonucleotide or saltthereof can have at least 90% sequence identity to any one of SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO:145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:201, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO:233, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO:243, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO:272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO:281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO:290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO:299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO:308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO:317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO:326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO:335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO:344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO:353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO:362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO:371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO:380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO:389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO:398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO:407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO:416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQID NO: 421, SEQ ID NO: 422, SEQ ID NO: 423, SEQ ID NO: 424, SEQ ID NO:425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO:434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO:443, SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO:453, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO:463, SEQ ID NO: 620, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO:651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO:660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO:669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO:678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO:687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO:696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 700, SEQID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO:705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO:714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO:723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, SEQ ID NO: 731, SEQ ID NO:732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO:741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO:750, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 753, SEQ ID NO: 754, SEQID NO: 755, SEQ ID NO: 756, SEQ ID NO: 757, SEQ ID NO: 758, SEQ ID NO:759, SEQ ID NO: 760, SEQ ID NO: 761, SEQ ID NO: 762, SEQ ID NO: 763, SEQID NO: 764, SEQ ID NO: 765, SEQ ID NO: 766, SEQ ID NO: 767, SEQ ID NO:768, SEQ ID NO: 769, SEQ ID NO: 770, SEQ ID NO: 771, SEQ ID NO: 772, SEQID NO: 773, SEQ ID NO: 774, SEQ ID NO: 775, SEQ ID NO: 776, SEQ ID NO:777, SEQ ID NO: 778, SEQ ID NO: 779, SEQ ID NO: 780, SEQ ID NO: 781, SEQID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, SEQ ID NO:786, SEQ ID NO: 787, SEQ ID NO: 788, SEQ ID NO: 789, SEQ ID NO: 790, SEQID NO: 791, SEQ ID NO: 792, SEQ ID NO: 793, SEQ ID NO: 794, SEQ ID NO:795, SEQ ID NO: 796, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQID NO: 800, SEQ ID NO: 801, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO:804, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 807, SEQ ID NO: 808, SEQID NO: 809, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812, SEQ ID NO:813, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 816, SEQ ID NO: 817, SEQID NO: 818, SEQ ID NO: 819, SEQ ID NO: 820, SEQ ID NO: 821, SEQ ID NO:822, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 825, SEQ ID NO: 835, SEQID NO: 836, SEQ ID NO: 837, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO:848, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851, SEQ ID NO: 852, SEQID NO: 853, SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO:857, SEQ ID NO: 858, SEQ ID NO: 859, SEQ ID NO: 860, SEQ ID NO: 861, SEQID NO: 862, SEQ ID NO: 863, SEQ ID NO: 864, SEQ ID NO: 865, SEQ ID NO:866, SEQ ID NO: 867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 870, SEQID NO: 871, SEQ ID NO: 872, SEQ ID NO: 873, SEQ ID NO: 874, SEQ ID NO:875, SEQ ID NO: 876, SEQ ID NO: 877, SEQ ID NO: 878, SEQ ID NO: 879, SEQID NO: 880, SEQ ID NO: 881, SEQ ID NO: 882, SEQ ID NO: 883, SEQ ID NO:884, SEQ ID NO: 885, SEQ ID NO: 886, SEQ ID NO: 887, SEQ ID NO: 888, SEQID NO: 889, SEQ ID NO: 890, SEQ ID NO: 891, SEQ ID NO: 892, SEQ ID NO:893, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896, SEQ ID NO: 897, SEQID NO: 898, or SEQ ID NO: 899, as determined by a BLAST pairwisesequence alignment algorithm. In some embodiments, an engineeredoligonucleotide sequence can comprise the sugar, base, or backbonemodification. In some embodiments, a modification can comprise a linker.In some embodiments, a linker can be a covalent linker. In someembodiments, a linker can be a cleavable linker. In some embodiments, alinker can be further modified to comprises a conjugate. In someembodiments, a conjugate can be an antibody, a naturally occurringligand, a small molecule, or a peptide. In some embodiments, a conjugatecan be a drug or salt thereof. In some embodiments, an engineeredoligonucleotide can comprise a base of a nucleotide that is glycosylatedwith a glycan. In some embodiments, the first or the second RNA can atleast partially comprise an mRNA sequence. In some embodiments, anengineered oligonucleotide or salt thereof, when contacted with the mRNAsequence, can produce at least about a 1.2-fold lower expression of apolypeptide encoded by the mRNA sequence, as compared to contacting anequivalent amount of the ncRNA with the mRNA sequence; as determined by:(a) transfecting the engineered oligonucleotide or salt thereof into afirst isolated mammalian cell comprising the mRNA sequence, (b)transfecting the ncRNA into a second isolated mammalian cell comprisingthe mRNA sequence, and (c) measuring an amount of the polypeptideexpressed in the first isolated mammalian cell and the second isolatedmammalian cell, wherein the first isolated mammalian cell and the secondisolated mammalian cell are of the same type of mammalian cell. In someembodiments, an engineered oligonucleotide or salt thereof, whencontacted with the mRNA sequence, can produce at least about a 1.2-foldlower activity of a polypeptide encoded by the mRNA sequence, ascompared to contacting an equivalent amount of the ncRNA with the mRNAsequence; as determined by: (a) transfecting the engineeredoligonucleotide or salt thereof into a first isolated mammalian cellcomprising the mRNA sequence, (b) transfecting the ncRNA into a secondisolated mammalian cell comprising the mRNA sequence, and (c) measuringan amount of activity from the polypeptide expressed in the firstisolated mammalian cell and the second isolated mammalian cell, whereinthe first isolated mammalian cell and the second isolated mammalian cellare of the same type of mammalian cell. In some embodiments, anengineered oligonucleotide or salt thereof when contacted with the mRNAsequence can produce from about 1.2-fold to about 10-fold lowerexpression of the polypeptide encoded by the mRNA sequence, as comparedto contacting the equivalent amount of the ncRNA; as determined by: (a)transfecting the engineered oligonucleotide or salt thereof into thefirst isolated mammalian cell comprising the mRNA sequence, (b)transfecting the ncRNA into the second isolated mammalian cellcomprising the mRNA sequence, and (c) measuring the amount of thepolypeptide expressed in the first isolated mammalian cell and thesecond isolated mammalian cell. In some embodiments, an engineeredoligonucleotide or salt thereof when contacted with the mRNA sequencecan produce from about 1.2-fold to about 10-fold lower activity of thepolypeptide encoded by the mRNA sequence, as compared to contacting theequivalent amount of the ncRNA; as determined by: (a) transfecting theengineered oligonucleotide or salt thereof into the first isolatedmammalian cell comprising the mRNA sequence, (b) transfecting the ncRNAinto the second isolated mammalian cell comprising the mRNA sequence,and (c) measuring the amount of activity from the polypeptide expressedin the first isolated mammalian cell and the second isolated mammaliancell. In some embodiments, a first isolated mammalian cell and secondisolated mammalian cell can be a human cell or a mouse cell. In someembodiments, a first isolated mammalian cell can be a human cell,wherein the human cell can be a cancer cell, a fibroblast, a leukocyte,an epithelial cell, a squamous cell, a myoblast, a muscle cell In someembodiments, at least about 80% of an initial amount of the engineeredoligonucleotide or salt thereof can remain when the engineeredoligonucleotide or salt thereof is stored in a closed container storedfor a time period of at least about 1 month at about 23 degrees Celsiuswith a relative atmospheric humidity of about 50%. In some embodiments,a time period can be from about 1 month to about 1 year. In someembodiments, a disease or condition can comprise a cancer. In someembodiments, at least a portion of the first or the second RNA can beencoded by an oncogene. In some embodiments, an oncogene can compriseABL1, ABL2, AKT1, AKT2, AKT3, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF,CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2, BBIT3,BBX6, DEK, EGFR, ELK4, ERBB2, ERBB3, E2F1, ZEB1, ETV4, ETV6, EVIl,EWSR1, FEV, FGFR1, FGFR1OP, FGR2, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS,IRF4, ITGA6, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET,MITF, MLL, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1,NUP214, PAX8, PDGFB, PIK3CA, PIM1, PLAGI, PPARG, PTPN11, RAF1, REL, RET,ROS1, SETDB1, SERPINE1, SMO, SS18, TCL1A, TET2, TFG, CDK6, ATG9A, TLX1,TPR, USP6, CSNK1G, KLF17, ARHGAP26, RAB1IFIPI, RBJ, SERBP1, CTBP1, CRKL,ITGA3, ITGAV, LAMC1, G6PC2, PPP2R5E or any combination thereof. In someembodiments, an oncogene can comprise ITGA6, BCL2, DEK, PLAGI, SERPINE1,MYCN, LMO2, PIM1, EGFR, IRS1, NT5E, GLDC, SOCS1, STAT1, LOX, PDGFRB,WNT5A, CD80, CCNA1, THBS2, IGF1R, AFAP1L2, CTHRC1, MET, FAP, ILlA, GJA1,MYBL2, or any combination thereof. In some embodiments, an engineeredoligonucleotide or salt thereof can be selective for an RNA sequenceencoding ITGA6, SERPINE1, EGFR, MDTH or any combination thereof, among aplurality of RNA sequences. In some embodiments, a disease or conditioncan comprise fibrosis. In some embodiments, at least a portion of thefirst or the second RNA can be encoded by a collagen super family gene,a platelet-derived growth factor gene, a TGF-β signaling gene, acollagen remodeling gene, an extracellular matrix remodeling gene, a Wntsignaling gene, a hepatoma-derived growth factor (HDGF) signaling gene,or any combination thereof. In some embodiments, at least a portion ofan first or the second RNA can be encoded by the collagen super familygene, wherein the collagen super family gene is selected from the groupconsisting of: COL1A1, COL11A1, COL2A1, COL5A3, COL5A2, COL4A4, COL21A1,COL7A1, COL9A1, COL19A1, COL5A1, COL22A1, COL8A1, COL4A2, COL6A2,COL24A1, COL4A3, COL4A6, COL25A1, COL16A1, COL15A1, and any combinationthereof. In some embodiments, at least a portion of the first or thesecond RNA is encoded by the platelet-derived growth factor gene,wherein the platelet-derived growth factor gene can be selected from thegroup consisting of: PDGFB, PDGFC, PDGFRB, and any combination thereof.In some embodiments, at least a portion of the first or the second RNAcan be encoded by the TGF-β signaling gene, wherein the TGF-β signalinggene is WISP1, TGFB2, or any combination thereof. In some embodiments,at least a portion of the first or the second RNA can be encoded by thecollagen remodeling gene, wherein the collagen remodeling gene is LOXL2.In some embodiments, at least a portion of the first or the second RNAcan be encoded by the extracellular matrix remodeling gene, wherein theextracellular matrix remodeling gene is selected from the groupconsisting of: COL1A1, COL11A1, COL2A1, COL5A3, COL5A2, COL4A4, COL21A1,COL7A1, COL9A1, COL19A1, COL5A1, COL22A1, COL8A1, COL4A2, COL6A2,COL24A1, COL4A3, COL4A6, COL25A1, COL16A1, COL15A1, LOXL2, Elastin, andany combination thereof. In some embodiments, at least a portion of thefirst or the second RNA can be encoded by the Wnt signaling gene,wherein the Wnt signaling gene comprises WISP1. In some embodiments, atleast a portion of the first or the second RNA can be encoded by theHDGF signaling gene, wherein the HDGF signaling gene comprises HDGF. Insome embodiments, the disease or condition can comprise a viralinfection. In some embodiments, the viral infection can be an HCVGenotype 1 infection. In some embodiments, at least a portion of thefirst or the second RNA can be encoded in an HCV Genotype 1 genome. Insome embodiments, the first or the second RNA can comprise at leastabout 90% sequence identity to SEQ ID NO: 587, SEQ ID NO: 588, or SEQ IDNO: 589, as determined by a BLAST pairwise sequence alignment algorithm.In some embodiments, the viral infection can be a coronavirus infection.In some embodiments, the coronavirus can be SARS-CoV-2. In someembodiments, at least a portion of the first or the second RNA can beencoded in a SARS-CoV-2 genome. In some embodiments, the first or thesecond RNA can comprise at least about 90% sequence identity to any oneof SEQ ID NO: 500 to SEQ ID NO: 531, SEQ ID NO: 829, SEQ ID NO: 830, orSEQ ID NO: 831. In some embodiments, the first or the second RNA cancomprise at least about 90% sequence identity to SEQ ID NO: 500, SEQ IDNO: 513, or SEQ ID NO: 518, as determined by a BLAST pairwise sequencealignment algorithm. In some embodiments, the coronavirus can beSARS-CoV. In some embodiments, at least a portion of the first or thesecond RNA can be encoded in a SARS-CoV genome. In some embodiments, thefirst or the second RNA can comprise at least about 90% sequenceidentity to any one of SEQ ID NO: 474 to SEQ ID NO: 499, SEQ ID NO: 826,SEQ ID NO: 827, or SEQ ID NO: 828. In some embodiments, the first or thesecond RNA can comprise at least about 90% sequence identity to SEQ IDNO: 476, SEQ ID NO: 481, or SEQ ID NO: 495, as determined by a BLASTpairwise sequence alignment algorithm. In some embodiments, thecoronavirus can be MERS-CoV. In some embodiments, at least a portion ofthe first or the second RNA can be encoded in a MERS-CoV genome. In someembodiments, the first or the second RNA can comprise at least about 90%sequence identity to any one of SEQ ID NO: 532 to SEQ ID NO: 554. Insome embodiments, the coronavirus can be CoV-HKU1. In some embodiments,at least a portion of the first or the second RNA can be encoded in aCoV-HKU1 genome. In some embodiments, the first or the second RNA cancomprise at least about 90% sequence identity to any one of SEQ ID NO:555 to SEQ ID NO: 586. In some embodiments, the viral infection can bean HIV infection. In some embodiments, at least a portion of the firstor the second RNA can be encoded by an HIV genome. In some embodiments,the first or the second RNA can comprise at least about 90% sequenceidentity to SEQ ID NO: 470, as determined by a BLAST pairwise sequencealignment algorithm. In some embodiments, the first or the second RNAcan comprise at least about 90% sequence identity to SEQ ID NO: 471, SEQID NO: 472, or SEQ ID NO: 473, as determined by a BLAST pairwisesequence alignment algorithm. In some embodiments, the disease orcondition can comprise a neuromuscular disorder including a musculardystrophy or a myopathy. In some embodiments, the disease or conditioncan be a Duchenne's muscular dystrophy (DMD), Myotonic Dystrophy (MD),Facioscapulohumeral muscular dystrophy (FSHD), Limb-Girdle musculardystrophy (LGMD), Becker muscular dystrophy, Oculopharyngeal musculardystrophy, Emery-Dreifuss muscular dystrophy, or Distal musculardystrophy. In some embodiments, the disease or condition can be causedby an inherited or spontaneous autosomal dominant mutation. In someembodiments, at least a portion of the first or the second RNA can beencoded in the dystrophin, DMPK, CLCN1, CNBP, D4Z4 repeat, DUX4, SMCHD1,DBET, SVIL, GAL3ST2, FRG1, CAPN3, DYSF, LMNA, PABPN1, PYGM, MYOD1, MYH7,HNRNPC, HNRNPA2B1, ACVR1, ASIC2, ATG14, ATP1A1, B3GTNL1, BANF1, BPTF,CASP8AP2, CDX4, CELF2,CHMP7, CKMT1B, CLASPI, CNOT3, COL15A1, CYP3A4,DCAF15, DCN, DLX5, DUSP7, DUX1, DUX5, EMILINI, EPG5, FAM13A, FBX03,FBXL22, FMNL3, FREM2, FRMPD2, GADD45A, GID4, GJD3, GMPR, GNAT1, GOSRI,GPRC6A, HERC1, HGF, HOOK3, HOXC9, HSP40, IRF9, IRX5, ITGA10, ITGA3,ITGA9, KCNC3, KLHL3, KLK6, LARP6, MALT1, MAP3K4, MAPK10, MIR4661,MIR8078, MTSS1, NDUFAF6, NEBL, NKX2, NR2F1, PCID2, PDE10A, PKD1L2,PKHD1, PPP1R12B, PTPRN2, PYY, RABGAP1L, RBCK1, RFX3, RHBDF2, SCRIB,SEMA3B, SETD4, SHFL, SHH, SLC37A4, SLC9A8, SMAD1, SPEF1, SPRED3,ST3GAL6, STAGI, SUPV3L1, TBC1D26, TCEA2, TCF3, TM6SF1, TMEM 108,TMEM259, TNFSF4, TNIP1, TRNP1, USH1G, WRNIP1, XIAP, ZNF574 gene or anycombination thereof. In some embodiments, a first or the second RNA cancomprise at least about 90% sequence identity to any one of SEQ ID NO:901 to SEQ ID NO: 949, as determined by a BLAST pairwise sequencealignment algorithm. In some embodiments, at least one base of anucleotide in the engineered oligonucleotide may not be complementary tothe first or the second RNA.

Also disclosed herein are engineered oligonucleotides or salts thereofthat can comprise a polynucleotide sequence of from about 5 nucleotidesto about 50 nucleotides. In some embodiments, the engineeredoligonucleotide or salt thereof can comprise: a first region, a secondregion that is adjacent to the first region, and a third region that isadjacent to the second region, wherein the regions are arranged from 5′to 3′ in the following order: the first region, the second region, andthe third region; wherein when the engineered oligonucleotide or saltthereof is bound to an mRNA sequence, the first region and the thirdregion are complementary to the mRNA sequence and the second regioncomprises at least one base that is not complementary to the mRNAsequence. In some embodiments, the engineered oligonucleotide or saltthereof can comprise an at least about 10% lower Gibbs free energy (ΔG)of binding, as determined for binding to the mRNA sequence at about 37degrees Celsius and at about pH 7.2, relative to a ΔG of binding of anotherwise comparable oligonucleotide binding to the mRNA sequence at 37degrees Celsius and at about pH 7.2, wherein the otherwise comparableoligonucleotide lacks the at least one base in the engineeredoligonucleotide that is not complementary to the mRNA sequence.

Also disclosed herein are engineered oligonucleotides or salts thereofthat can comprise a polynucleotide sequence with at least about 90%sequence identity to any one of SEQ ID NOs: 1-5, 12-14, 19-20, 24-25,28, 30, 32, 34, 36, 38-45, 52-89, 100-154, 184-201, 205-222, 225-233,235-243, 245-443, 445-453, 455-463, 465, 620, 624-825, 835-837, 840-899,and 901-949, as determined by a BLAST pairwise sequence alignmentalgorithm, wherein the engineered oligonucleotide or salt thereof whencontacted with an mRNA sequence produces at least about a 1.2-fold lowerexpression of a polypeptide encoded by the mRNA sequence, as compared tocontacting an equivalent amount of an miR-29 or miR-30 oligonucleotideor salt thereof naturally present in a human cell; as determined by: (a)transfecting the engineered oligonucleotide or salt thereof into a firstisolated human cell comprising the mRNA sequence, (b) transfecting themiR-29 or miR-30 oligonucleotide into a second isolated human cellcomprising the mRNA sequence, and (c) measuring an amount of thepolypeptide expressed in the first isolated human cell and the isolatedsecond human cell. In some embodiments, the structure and chemistry canbe optimized to impart greater than or equal to 100X stability tonatural nucleases compared to an unmodified sequence or a comparablencRNA.

Also disclosed herein are engineered passenger oligonucleotides or saltsthereof that can comprise a polynucleotide sequence, wherein theengineered passenger oligonucleotide or salt thereof can be at leastpartially complementary to at least a portion of an engineeredoligonucleotide or salt thereof as described herein. In someembodiments, an engineered passenger oligonucleotide or salt thereof canbe from about 5 to about 50 nucleotides in length. In some embodiments,the engineered passenger oligonucleotide or salt thereof can comprise aribose sugar. In some embodiments, the engineered passengeroligonucleotide or salt thereof can comprise a deoxyribose sugar. Insome embodiments, the engineered passenger oligonucleotide or saltthereof can have at least 90% sequence identity to any one of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO:162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, or SEQ ID NO: 466, asdetermined by a BLAST pairwise sequence alignment algorithm. In someembodiments, the engineered passenger oligonucleotide can form asecondary structure comprising a stem-loop. In some embodiments, theengineered passenger oligonucleotide or salt thereof can comprise achemically modified base, chemically modified sugar, chemically modifiedbackbone or phosphate linkage, or any combination thereof relative to anaturally occurring base, sugar, backbone, or phosphate linkage. In someembodiments, the chemical modification can be selected from the groupconsisting of: a methyl group, a fluoro group, a methoxyethyl group, anethyl group, a hydroxymethyl group, a formyl group, a carboxylic acid orsalt thereof, a phosphothionate modified backbone, a methylphosphonatemodified backbone, an amino-alkyl chain modification, and anycombination thereof. In some embodiments, the engineered passengeroligonucleotide or salt thereof, when chemically modified, can comprisethe formula: CAP-mNmNmN(N)kmNmNmN; wherein CAP can be selected from a5′-terminal methyl group (5′-OMethyl) or alkylamino group such asamino-carbon 6 chain (5′-Amino C6); each N can be independently uracil,guanine, adenine, or cytosine; each mN can be independently a2′-O-methyl-modified uracil, guanine, adenine, or cytosine; and each kcan be from 12-19. In some embodiments, the engineered passengeroligonucleotide or salt thereof can have at least 90% sequence identityto any one of SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO:93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ IDNO: 98, SEQ ID NO: 99, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172,SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ IDNO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181,SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 202, SEQ ID NO: 203, SEQ IDNO: 204, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 234, SEQ ID NO: 244,SEQ ID NO: 444, SEQ ID NO: 454, SEQ ID NO: 464, SEQ ID NO: 838, SEQ IDNO: 839, or SEQ ID NO: 900, as determined by a BLAST pairwise sequencealignment algorithm. In some embodiments, the engineered passengeroligonucleotide or salt thereof can comprise a sugar modification. Insome embodiments, the sugar modification can comprise a glycosylatedbase. In some embodiments, the structure and chemistry can be optimizedto impart greater than or equal to 100X stability to natural nucleasescompared to an unmodified sequence or a comparable ncRNA.

Also disclosed herein are nucleic acid constructs that can comprise: (a)a first strand comprising an engineered oligonucleotide or salt thereofas described herein and (b) a second strand comprising an engineeredpassenger oligonucleotide or salt thereof as described herein with asequence complementary to at least a portion of the first strand.

Also disclosed herein are vectors that can comprise an engineeredoligonucleotide or salt thereof as described herein or a nucleic acidconstruct as described herein. In some embodiments, the vector can bepresent in a liposome, a nanoparticle, or any combination thereof. Insome embodiments, the vector can be a viral vector. In some embodiments,the viral vector can be an adeno-associated viral (AAV) vector.

Also disclosed herein are isolated cells that can comprise an engineeredoligonucleotide or salt as described herein, a nucleic acid construct asdescribed herein, or a vector as described herein.

Also disclosed herein are pharmaceutical compositions that can comprise:(a) an engineered oligonucleotide or salt thereof as described herein, anucleic acid construct as described herein, or a vector as describedherein; and (b) a pharmaceutically acceptable excipient, diluent, orcarrier. In some embodiments, a pharmaceutical composition can be inunit dose form. In some embodiments, a pharmaceutical composition can beencapsulated. In some embodiments, a pharmaceutical composition can bein the form of a liquid.

Also disclosed herein are methods of treating a subject in need thereofthat can comprise: administering to the subject a therapeuticallyeffective amount of: an engineered oligonucleotide or salt thereof asdescribed herein, a nucleic acid construct as described herein, a vectoras described herein, or a pharmaceutical composition as describedherein. In some embodiments, the administering can be by an intravenousinjection, an intramuscular injection, an intrathecal injection, anintraorbital injection, a subcutaneous injection, or any combinationthereof. In some embodiments, the administering can be oral, otic,ocular, rectal, or any combination thereof. In some embodiments, themethod can further comprise a second administering comprising a secondtherapy to the subject. In some embodiments, the administering and thesecond administering can be concurrent. In some embodiments, theadministering and the second administering can be sequential. In someembodiments, the subject can have or can be at risk of developing adisease or condition. In some embodiments, the disease or condition canbe a cancer. In some embodiments, the cancer can be a head cancer, aneck cancer, skin cancer, a cervical cancer, a prostate cancer, or anycombination thereof. In some embodiments, the disease or condition canbe a viral infection. In some embodiments, the viral infection can be aSARS-CoV infection, a SARS-COV-2 infection, a MERS-CoV infection, aCoV-HKU1 infection, an HIV infection, or an HCV infection. In someembodiments, the disease or condition can be a fibrosis. In someembodiments, the disease or condition can be muscular dystrophy. In someembodiments, the subject can be a mammal. In some embodiments, themammal can be a human. In some embodiments, the subject may have beendiagnosed with a disease or condition by a diagnostic test. In someembodiments, a diagnostic test can comprise an imaging procedure, ablood count analysis, a tissue pathology analysis, a biomarker analysis,or any combination thereof.

Also disclosed herein are methods that can comprise: contacting anengineered oligonucleotide or salt thereof as described herein, anucleic acid construct as described herein, or a vector as describedherein, with an isolated cell or an isolated tissue.

Also disclosed herein are as described herein kits that can comprise anengineered oligonucleotide or salt thereof as described herein in acontainer, a nucleic acid construct as described herein in a container,a vector as described herein in a container, or a pharmaceuticalcomposition as described herein in a container.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

DESCRIPTION OF THE DRAWINGS

The novel features of exemplary embodiments are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of exemplary embodiments are utilized, and theaccompanying drawings of which:

FIG. 1A shows natural miR-30 guide strand sequences and examples ofengineered family members.

FIG. 1B shows natural miR-30 passenger strand sequences and examples ofengineered family members.

FIG. 2A shows free energy (ΔG) and hybridization between naturalmiR-30a-5p sequence and target sites in the 3′UTRs of ITGA6, SERPINE1,and EGFR transcripts.

FIG. 2B shows free energy (ΔG) and hybridization between engineeredfamily G007-30 (SEQ ID NO: 39) containing the ‘G’ insertion at position13 and the ITGA6, SERPINE1, and EGFR target sites.

FIG. 2C shows that a mimic containing the same ‘G’ insertion at position13 as found in G007-30 can demonstrate improved knockdown of selectoncogenic targets.

FIG. 3 shows a schematic visualization of miR-30-5p target sites in theMTDH 3′UTR.

FIG. 4A shows anti-tumor activity of miR-30 mimics in cancer cell lines.

FIG. 4B shows knock-down of a luciferase reporter containingcancer-relevant miR-30 target sites with its 3′ UTR by miR-30 mimics.

FIG. 4C shows the anti-tumor activity of native miR-30a and exemplarengineered mimics in additional cell lines with different geneticbackgrounds.

FIG. 4D shows anti-tumor activity of a panel of engineered miR-30 mimicsin additional cancel cell lines with different histology and geneticbackgrounds.

FIG. 5A shows an example of Urea-PAGE resolving a nucleic acid marker(M) and sample comprising mimic duplex, guide strand, and degradedfragments (≤n-1)

FIG. 5B shows duplex stability of engineered miR-30 miRNA mimics inhuman serum.

FIG. 6A shows structure of engineered mimic duplexes with passengerstrands sequence and chemical modifications which alter the mimicstructure.

FIG. 6B shows exemplar duplex stability of engineered miR-30 miRNAmimics in human serum.

FIG. 7A shows exemplar structure of engineered mimic duplexes withpassenger strands sequence and chemical modifications which alter themimic structure.

FIG. 7B shows anti-tumor activity of exemplar engineered miR-30 mimicsin cancer cell lines.

FIG. 7C shows knock-down of a luciferase reporter by engineered miR-30mimics with varied structure but identical guide strands.

FIG. 8A shows the structure of two blunting modifications that can bemade to the 5′ terminus of the guide strand. FIG. 8B shows bluntedanti-tumor activity of mimics with 5′ modified guide strands.

FIG. 9A shows structure of exemplar engineered miR-30 mimic duplexes.

FIG. 9B shows anti-tumor activity of exemplar engineered miR-30 mimicsin cancer cell lines.

FIG. 9C shows knock-down of a luciferase reporter by exemplar engineeredmiR-30 mimics with varied structure but identical guide strands.

FIG. 10A displays improved anticancer activity and silencing of exemplarengineered mimic M30-043. FIG. 10B shows knockdown of luciferaseactivity by exemplar engineered mimic M30-043.

FIG. 11A shows reductions in innate TNFα production after exposure ofPBMCs to engineered miR-30 mimics

FIG. 11B shows reductions in innate IFNα production after exposure ofPBMCs to engineered miR-30 mimics.

FIG. 12A shows production of cisplatin resistant cancer cell lines.

FIG. 12B shows preservation of engineered miR-30 mimic activity incisplatin resistant cancer cell lines.

FIG. 12C shows resensitization of resistance cell lines to cisplatin byengineered miR-30 mimic treatment.

FIG. 12D shows sensitization of cancer cell lines to EGFR inhibitorcetuximab by engineered miR-30 mimic treatment.

FIG. 13A shows luciferase images of anti-tumor activity in orthotopicHNSCC tumors by engineered miR-30 mimics. FIG. 13B shows quantificationof luciferase activity in orthotopic HNSCC tumors by engineered miR-30mimics.

FIG. 13C shows anti-tumor activity of engineered miR-30 mimic M30-040 ina subcutaneous HNSCC mouse model.

FIG. 14A shows luciferase images of anti-tumor activity in orthotopicHNSCC tumors by engineered miR-30 mimic M30-048 with local treatment.FIG. 14B shows quantification of luciferase activity in orthotopic HNSCCtumors by engineered miR-30 mimic M30-048 with local treatment.

FIG. 15 shows natural miR-29 guide strand sequences and examples ofengineered family members.

FIG. 16 shows a schematic visualization of a human host cell gene TET-1and improved predicted binding by engineered miR-29 mimics.

FIG. 17 figure shows that engineered miR-29 mimics can have equivalentor greater knockdown activity compared to native miR-29b in a luciferasecell line.

FIG. 18A shows sequence and structure of native miR-29b-1 duplex andengineered miR-29 mimics.

FIG. 18B shows duplex stability of native and engineered miR-29 miRNAmimics in human serum.

FIG. 19A shows sequence and structure of exemplar engineered miR-29mimics.

FIG. 19B shows duplex stability of exemplar engineered miR-29 miRNAmimics in human serum.

FIG. 20A shows sequence and structure of native miR-29a duplex andexemplar engineered miR-29a mimics.

FIG. 20B shows sequence and structure of native miR-29b duplex andexemplar engineered miR-29b mimics.

FIG. 20C shows improvement of luciferase knockdown activity by exemplarengineered mimics of miR-29a and miR-29b.

FIG. 21 shows engineered mimics of miR-29 display reduced innate immunestimulation.

FIG. 22A shows schematic visualization of an native and engineeredmiR-29-3p guide strands binding to HIV-1 NEF RNA.

FIG. 22B shows inhibition of HIV-1 replication by exemplar engineeredmiR-29 mimics.

FIG. 22C shows decreased GFP reporter expression by M29-012 and M29-028mimics.

FIG. 23A, FIG. 23B and FIG. 23C shows improved knockdown of HIV-1relevant host mRNAs by engineered mimics of miR-29.

FIG. 24A shows calculated free energy of binding to predicted targetsites in the SARS-CoV viral genome by natural and engineered miR-29-3pguide strands.

FIG. 24B shows calculated free energy of binding to predicted targetsites in the SARS-CoV viral genome by natural and engineered miR-30-5pguide strands.

FIG. 25A shows calculated free energy of binding to predicted targetsites in the SARS-CoV-2 viral genome by natural and engineered miR-29-3pguide strands.

FIG. 25B shows calculated free energy of binding to predicted targetsites in the SARS-CoV-2 viral genome by natural and engineered miR-30-5pguide strands.

FIG. 25C shows inhibition of SARS-CoV-2 viral replication by exemplarengineered miR-29 and miR-30 mimics.

FIG. 26 shows calculated free energy of binding to predicted targetsites in the HCV-1 viral genome by natural and engineered miR-29-3pguide strands.

FIG. 27 shows cell killing activity of anti-cancer engineered artificialmiRNA mimic ENG-miR-1 in normal and cancer cell lines.

FIG. 28 shows simultaneous knockdown of DUX4 and DBET RNA transcripts inFSHD patient myoblasts by multi-targeted ASOs.

FIG. 29 is a diagram showing a method and system as disclosed herein.

FIG. 30 shows a computer control system that is programmed or otherwiseconfigured to implement methods provided herein.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying Sequence Listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. 1.822.

SEQ ID NOs: 1-37 are the nucleotide sequences of exemplary maturemiRNAs.

SEQ ID NOs: 38-45, 52-89, and 624-825 are modified miR-30 guide strandnucleotide sequences.

SEQ ID NOs: 46-51, 90-99, and 900 are modified miR-30 passenger strandnucleotide sequences.

SEQ ID NOs: 100-154, 620, and 835-837 are modified miR-29 guide strandnucleotide sequences.

SEQ ID NOs: 155-183 and 838-839 are modified miR-29 passenger strandnucleotide sequences.

SEQ ID NOs: 184-201 are modified miR-26 guide strand nucleotidesequences.

SEQ ID NOs: 202-204 are modified miR-26 passenger strand nucleotidesequences.

SEQ ID NOs: 205-222 are modified miR-27 guide strand nucleotidesequences.

SEQ ID NOs: 223-224 are modified miR-27 passenger strand nucleotidesequences.

SEQ ID NOs: 225-233 are modified miR-101 guide strand nucleotidesequences.

SEQ ID NOs: 234 is a modified miR-101 passenger strand nucleotidesequence.

SEQ ID NOs: 235-243 are modified miR-145 guide strand nucleotidesequences.

SEQ ID NOs: 244 is a modified miR-145 passenger strand nucleotidesequence.

SEQ ID NOs: 245-443 and 840-899 are modified miR-205 guide strandnucleotide sequences.

SEQ ID NOs: 444 is a modified miR-205 passenger strand nucleotidesequence.

SEQ ID NOs: 445-453 are modified miR-338 guide strand nucleotidesequences.

SEQ ID NOs: 454 is a modified miR-338 passenger strand nucleotidesequence.

SEQ ID NOs: 455-463 are modified miR-375 guide strand nucleotidesequences.

SEQ ID NOs: 464 is a modified miR-375 passenger strand nucleotidesequence.

SEQ ID NOs: 465 is an engineered guide strand nucleotide sequence.

SEQ ID NOs: 466 is an engineered passenger strand nucleotide sequence.

SEQ ID NOs: 901-949 are antisense nucleotide sequences.

SEQ ID NO: 467-619, 621-623, 826-834, 950-1120 are target nucleotidesequence.

DETAILED DESCRIPTION Definitions

Unless otherwise indicated, open terms for example “contain,”“containing,” “include,” “including,” and the like mean comprising.

The singular forms “a”, “an”, and “the” are used herein to includeplural references unless the context clearly dictates otherwise.Accordingly, unless the contrary is indicated, the numerical parametersset forth in this application are approximations that can vary dependingupon the desired properties sought to be obtained.

As used herein, the term “about” or “approximately” can mean within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, e.g., the limitations of the measurement system.For example, “about” can mean plus or minus 10%, per the practice in theart. Alternatively, “about” can mean a range of plus or minus 20%, plusor minus 10%, plus or minus 5%, or plus or minus 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, within5-fold, or within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed. Also, where ranges and/or subranges of valuesare provided, the ranges and/or subranges can include the endpoints ofthe ranges and/or subranges.

The term “substantially” as used herein can refer to a value approaching100% of a given value. In some cases, the term can refer to an amountthat can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 99.99% of a total amount. In some cases, the term canrefer to an amount that can be about 100% of a total amount.

The term “homology” can refer to a % identity of a sequence to areference sequence. As a practical matter, whether any particularsequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%,97%, 98% or 99% identical to any sequence described herein (which cancorrespond with a particular nucleic acid sequence described herein),such particular polypeptide sequence can be determined conventionallyusing known computer programs such the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711). Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence, the parameters can be set such that the percentageof identity is calculated over the full length of the reference sequenceand that gaps in homology of up to 5% of the total reference sequenceare allowed.

For example, in a specific embodiment the identity between a referencesequence (query sequence, i.e., a sequence as described herein) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some cases,parameters for a particular embodiment in which identity is narrowlyconstrued, used in a FASTDB amino acid alignment, can include: ScoringScheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject sequence,whichever is shorter. According to this embodiment, if the subjectsequence is shorter than the query sequence due to N- or C-terminaldeletions, not because of internal deletions, a manual correction can bemade to the results to take into consideration the fact that the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity can be corrected by calculating thenumber of residues of the query sequence that are lateral to the N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedcan be determined by results of the FASTDB sequence alignment. Thispercentage can be then subtracted from the percent identity, calculatedby the FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score can beused for the purposes of this embodiment. In some cases, only residuesto the N- and C-termini of the subject sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryresidue positions outside the farthest N- and C-terminal residues of thesubject sequence are considered for this manual correction. For example,a 90 residue subject sequence can be aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for.

In some cases, an engineered oligonucleotide or salt thereof cancomprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% sequence identity to an oligonucleotide of any SEQ ID NO asdescribed herein. In some cases, an engineered oligonucleotide or saltthereof can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% sequence identity to at least 10 contiguous bases of anoligonucleotide of any one of SEQ ID NOs as described herein.

TABLE 1 Natural Human miRNAs SEQ ID miRNA Oligo Sequence (5′-3′) NO:miR-30a-5p UGUAAACAUCCUCGACUGGAAG  1 miR-30b-5p UGUAAACAUCCUACACUCAGCU 2 miR-30c-5p UGUAAACAUCCUACACUCUCAGC  3 miR-30d-5pUGUAAACAUCCCCGACUGGAAG  4 miR-30e-5p UGUAAACAUCCUUGACUGGAAG  5miR-30a-3p CUUUCAGUCGGAUGUUUGCAGC  6 miR-30b-3p CUGGGAGGUGGAUGUUUACUUC 7 miR-30c-1-3p CUGGGAGAGGGUUGUUUACUCC  8 miR-30c-2-3pCUGGGAGAAGGCUGUUUACUCU  9 miR-30d-3p CUUUCAGUCAGAUGUUUGCUGC 10miR-30e-3p CUUUCAGUCGGAUGUUUACAGC 11 miR-29a-3p UAGCACCAUCUGAAAUCGGUUA12 miR-29b-3p UAGCACCAUUUGAAAUCAGUGUU 13 miR-29c-3pUAGCACCAUUUGAAAUCGGUUA 14 miR-29a-5p ACUGAUUUCUUUUGGUGUUCAG 15miR-29b-1-5p GCUGGUUUCAUAUGGUGGUUUAGA 16 miR-29b-2-5pCUGGUUUCACAUGGUGGCUUAG 17 miR-29c-5p UGACCGAUUUCUCCUGGUGUUC 18miR-26a-5p UUCAAGUAAUCCAGGAUAGGCU 19 miR-26b-5p UUCAAGUAAUUCAGGAUAGGU 20miR-26a-1-3p CCUAUUCUUGGUUACUUGCACG 21 miR-26a-2-3pCCUAUUCUUGAUUACUUGUUUC 22 miR-26b-3p CCUGUUCUCCAUUACUUGGCUC 23miR-27a-5p AGGGCUUAGCUGCUUGUGAGCA 24 miR-27b-5p AGAGCUUAGCUGAUUGGUGAAC25 miR-27a-3p UUCACAGUGGCUAAGUUCCGC 26 miR-27b-3p UUCACAGUGGCUAAGUUCUGC27 miR-101-3p UACAGUACUGUGAUAACUGAA 28 miR-101-5p CAGUUAUCACAGUGCUGAUGCU29 miR-145-5p GUCCAGUUUUCCCAGGAAUCCCU 30 miR-145-3pGGAUUCCUGGAAAUACUGUUCU 31 miR-205-5p UCCUUCAUUCCACCGGAGUCUG 32miR-205-3p GAUUUCAGUGGAGUGAAGUUC 33 miR-338-3p UCCAGCAUCAGUGAUUUUGUUG 34miR-338-5p AACAAUAUCCUGGUGCUGAGUG 35 miR-375-3p UUUGUUCGUUCGGCUCGCGUGA36 miR-375-5p GCGACGAGCCCCUCGCACAAACC 37

TABLE 2 natural miRNA mimic composition miRNA Name Guide StrandPassenger Strand miR-30a miR-30a-5p (SEQ ID NO. 1) miR-30a-3p (SEQ IDNO. 6) miR-30b miR-30b-5p (SEQ ID NO. 2) miR-30b-3p (SEQ ID NO. 7)miR-30c-1 miR-30c-5p (SEQ ID NO. 3) miR-30c-1-3p (SEQ ID NO. 8)miR-30c-2 miR-30c-5p (SEQ ID NO. 3) miR-30c-2-3p (SEQ ID NO. 9) miR-30dmiR-30d-5p (SEQ ID NO. 4) miR-30d-3p (SEQ ID NO. 10) miR-30e miR-30e-5p(SEQ ID NO. 5) miR-30e-3p (SEQ ID NO. 11) miR-29a miR-29a-3p (SEQ ID NO.12) miR-29a-5p (SEQ ID NO. 15) miR-29b-1 miR-29b-3p (SEQ ID NO. 13)miR-296-1-5p (SEQ ID NO. 16) miR-29b-2 miR-29b-3p (SEQ ID NO. 13)miR-296-2-5p (SEQ ID NO. 17) miR-29c miR-29c-3p (SEQ ID NO. 14)miR-29c-5p (SEQ ID NO. 18) miR-26a-1 miR-26a-5p (SEQ ID NO. 19)miR-26a-1-3p (SEQ ID NO. 21) miR-26a-2 miR-26a-5p (SEQ ID NO. 19)miR-26a-2-3p (SEQ ID NO. 22) miR-26b miR-26b-5p (SEQ ID NO. 20)miR-26b-3p (SEQ ID NO. 23) miR-27a miR-27a-5p (SEQ ID NO. 24) miR-27a-3p(SEQ ID NO. 26) miR-27b miR-27b-5p (SEQ ID NO. 25) miR-27b-3p (SEQ IDNO. 27) miR-101 miR-101-3p (SEQ ID NO. 28) miR-101-5p (SEQ ID NO. 29)miR-145 miR-145-5p (SEQ ID NO. 30) miR-145-3p (SEQ ID NO. 31) miR-205miR-205-5p (SEQ ID NO. 32) miR-205-3p (SEQ ID NO. 33) miR-338 miR-338-3p(SEQ ID NO. 34) miR-338-5p (SEQ ID NO. 35) miR-375 miR-375-3p (SEQ IDNO. 36) miR-375-5p (SEQ ID NO. 37)

TABLE 3 Engineered miRNAs SEQ ID Oligo Sequence (5′-3′) NO: G006-30UGUAAACAUCCUGCGACUGGAA  38 G007-30 UGUAAACAUCCUGCGACUGGAAG  39 G061-30UGUAAACAUCCCGCGACUGGAAG  40 G062-30 UGUAAACAUCCUGUGACUGGAAG  41 G063-30UGUAAACAUCCCGUGACUGGAAG  42 G064-30 UGUAAACAUCCUGACACUCUCAGC  43 G065-30UGUAAACAUCCUGACACUCUCAG  44 G066-30 UGUAAACAUCCUGACACUCUCA  45 G075-30UGUAAACAUCCUGACACUCAGCU 624 G076-30 UGUAAACAUCCCGUGACAGGAAG 625 G077-30UGUAAACAUCCUGCGACUAGGAA 626 G078-30 UGUAAACAUCCUGCGACUGGUAA 627 G079-30UGUAAACAUCCCUGACUGGAAG 628 G080-30 UGUAAACAUCCUCGACUCAGCU 629 G081-30UGUAAACAUCCUCGACUCUCAGC 630 G082-30 UGUAAACAUCCUUCACUCAGCU 631 G083-30UGUAAACAUCCUUCACUCUCAGC 632 G084-30 UGUAAACAUCCCACACUCAGCU 633 G085-30UGUAAACAUCCCACACUCUCAGC 634 G086-30 UGUAAACAUCCCUCACUCAGCU 635 G087-30UGUAAACAUCCCUCACUCUCAGC 636 G088-30 UGUAAACAUCCUGCGACUCAGCU 637 G089-30UGUAAACAUCCUCGACAGGAAG 638 G090-30 UGUAAACAUCCUGCGACAGGAAG 639 G091-30UGUAAACAUCCUCGACAUGGAAG 640 G092-30 UGUAAACAUCCUCGAACUGGAAG 641 G093-30UGUAAACAUCCUGCGAACUGGAA 642 G094-30 UGUAAACAUCCCUGACAGGAAG 643 P001-30CCAGUCGAGGAUGUUUACA  46 P002-30 CCAGUCGCAGGAUGUUUACA  47 P003-30UCCAGUCGAGGAUGUUUACA  48 P004-30 UCCAGUCGCAGGAUGUUUACA  49 P005-30UCCAGUCGGAUGUUUACA  50 P125-30 UUCAGUCGGAUGUUUGCAGC  51 G008-30fUmGfUmAfAmAfCmAfUmCfCmUmGfCmGfAmCfUmGfGmAdAy  52 mG G009-30fUmGfUmAfAmAfCmAfUmCfCmUmGfCmGfAmCfUmGfGmAfAy  53 mG G010-30fUmGfUmAfAmAfCmAfUmCfCmUmGsfCmGfAmCfUmGfGmAsfAs  54 mG G011-30UGUAAACAUCmCmUmCmGmAmCmUmGGAAG  55 G012-30fUmGfUmAfAmAfCmAfUmCfCmUmGyfCmGfAmCfUmGfGmAsf  56 AsmG G014-30fUmGfUmAfAmAfCmAfUmCfCmUsmGfCmGfAmCfUmGfGmAsfAs  57 mG G025-30fUmGfUmAfAmAfCmAfUmCfCmUsmGsfCmGfAmCfUmGfGmAsf  58 AsmG G026-30fUmGfUmAfAmAfCmAfUmCfCmUymGfCmGfAmCfUmGfGmAsf  59 AsmG G027-30UGUAAACAUCmCmUmGmCmGmAmCmUGGfAymA  60 G028-30UGUAAACAUCmCmUmGmCmGmAmCmUGGsfAsmA  61 G029-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGAfAymG  62 G030-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGfAymA  63 G031-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGsfAsmA  64 G032-30fUmGfUmAfAmAfCmAfUmCfCmUmGfCmGfAmCfUmGfGmAsfAs  65 mG G033-30UGUAAACAUCmCmUmGsmCmGmAmCmUmGGAsfAsmA 644 G034-30UGUAAACAUCmCmUmGymCmGmAmCmUmGGAsfAsmA  66 G035-30mUsmGUAAACAUCmCmUmGmCmGmAmCmUmGGAsfAsmG  67 G038-30UGUAAACAUCmCmUsmGmCmGmAmCmUmGGsfAsmA 645 G039-30fUmGfUAAACAfUmCfCmUfCmGfAmCfUmGfGmAsfAsmG  68 G042-30fUmGfUmAfAmAfCmAfUmCfCmUfCmGfAmCfUmGfGmAsfAsmG  69 G056-30UGUAAACAUCmCmUsmGsmCmGmAmCmUmGGsfAsmA  70 G057-30UGUAAACAUCmCmUymGmCmGmAmCmUmGGsfAsmA  71 G058-30UGUAAACAUfCmCfUmGfCmGfAmCfUGGsfAsmA  72 G059-30UGUAAACAUmCfCmUfGmCfGmAfCmUGGsfAsmA  73 G060-30UGUAAACAUmCmCmUmGmCmGmAmCmUGGsfAsmA  74 G067-30UGUAAACAUCmCmUmsGmCmGmAmCmUmGGsfAsmA  75 G068-30UGUAAACAUCmCmUmGmsCmGmAmCmUmGGAsfAsmA  76 G069-30UGUAAACAUCmCmUmAmCmAmCmUmCAGsfCsmU  77 G070-30UGUAAACAUCmCmCmCmGmAmCmUmGGAsfAsmG  78 G071-30UGUAAACAUCmCmCmGmCmGmAmCmUmGGAsfAsmG  79 G072-30mUsmGUAAACAUCmCmUmGmCmGmAmCmUmGGmAsfAsmG  80 G073-30mUsmGUAAACAUmCmCmUmGmCmGmAmCmUGGmAsfAsmG  81 G074-30mUsmGsmUAAACAUCmCmUmGmCmGmAmCmUmGGmAsfAsmG  82 G095-30UGUAAACAUCmCmCmUmGmAmCmAmGGApfApmG 646 G096-30UGUAAACAUCmCmCmUmGmAmCmAGGApfApmG 647 G097-30UGUAAACAUCmCmCmUmGmAmCAGGApfApmG 648 G098-30UGUAAACAUmCmCmCmUmGmAmCmAmGGApfApmG 649 G099-30UGUAAACAUmCmCmCmUmGmAmCmAGGApfApmG 650 G100-30UGUAAACAUmCmCmCmUmGmAmCAGGApfApmG 651 G101-30UGUAAACAmUmCmCmCmUmGmAmCmAmGGApfApmG 652 G102-30UGUAAACAmUmCmCmCmUmGmAmCmAGGApfApmG 653 G103-30UGUAAACAmUmCmCmCmUmGmAmCAGGApfApmG 654 G104-30UGUAAACAUCmCmUmGmCmGmAmAmCmUGGpfApmA 655 G105-30UGUAAACAUCmCmUmGmCmGmAmAmCUGGpfApmA 656 G106-30UGUAAACAUCmCmUmGmCmGmAmACUGGpfApmA 657 G107-30UGUAAACAUmCmCmUmGmCmGmAmAmCmUGGpfApmA 658 G108-30UGUAAACAUmCmCmUmGmCmGmAmAmCUGGpfApmA 659 G109-30UGUAAACAUmCmCmUmGmCmGmAmACUGGpfApmA 660 G110-30UGUAAACAmUmCmCmUmGmCmGmAmAmCmUGGpfApmA 661 G111-30UGUAAACAmUmCmCmUmGmCmGmAmAmCUGGpfApmA 662 G112-30UGUAAACAmUmCmCmUmGmCmGmAmACUGGpfApmA 663 G113-30UGUAAACAUCmCmUmCmGmAmAmCmUmGGApfApmG 664 G114-30UGUAAACAUCmCmUmCmGmAmAmCmUGGApfApmG 665 G115-30UGUAAACAUCmCmUmCmGmAmAmCUGGApfApmG 666 G116-30UGUAAACAUmCmCmUmCmGmAmAmCmUmGGApfApmG 667 G117-30UGUAAACAUmCmCmUmCmGmAmAmCmUGGApfApmG 668 G118-30UGUAAACAUmCmCmUmCmGmAmAmCUGGApfApmG 669 G119-30UGUAAACAmUmCmCmUmCmGmAmAmCmUmGGApfApmG 670 G120-30UGUAAACAmUmCmCmUmCmGmAmAmCmUGGApfApmG 671 G121-30fUmGfUmAfAmAfCmAfUmCfCmUfCmGfAmCfUmGfGmAdAymG  83 G122-30fUmGfUAAACAfUmCfCmUfCGACUGfGmAsfAsmG  84 G123-30UGUAAACAmUmCmCmUmCmGmAmAmCUGGApfApmG 672 G124-30UGUAAACAUCmCmUmCmGmAmCmAmUmGGApfApmG 673 G125-30UGUAAACAUCmCmUmCmGmAmCmAmUGGApfApmG 674 G126-30UGUAAACAUCmCmUmCmGmAmCmAUGGApfApmG 675 G127-30UGUAAACAUmCmCmUmCmGmAmCmAmUmGGApfApmG 676 G128-30 OMe-  85dTmGfUmAfAmAfCmAfUmCfCmUmGfCmGfAmCfUmGfGmAsfAs mG G129-30UGUAAACAUCmCmUmCmGmAmCmUmGGAsfAsmG  86 G130-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGAAG  87 G131-30UGUAAACAUmCmCmUmCmGmAmCmAmUGGApfApmG 677 G132-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGAsfAsmG  88 G133-30UGUAAACAUCmCmUmGmCmGmAmCmUmGGAA  89 G134-30UGUAAACAUmCmCmUmCmGmAmCmAUGGApfApmG 678 G135-30UGUAAACAmUmCmCmUmCmGmAmCmAmUmGGApfApmG 679 G136-30UGUAAACAmUmCmCmUmCmGmAmCmAmUGGApfApmG 680 G137-30UGUAAACAmUmCmCmUmCmGmAmCmAUGGApfApmG 681 G138-30UGUAAACAUCmCmUmGmCmGmAmCmAmGGApfApmG 682 G139-30UGUAAACAUCmCmUmGmCmGmAmCmAGGApfApmG 683 G140-30UGUAAACAUCmCmUmGmCmGmAmCAGGApfApmG 684 G141-30UGUAAACAUmCmCmUmGmCmGmAmCmAmGGApfApmG 685 G142-30UGUAAACAUmCmCmUmGmCmGmAmCmAGGApfApmG 686 G143-30UGUAAACAUmCmCmUmGmCmGmAmCAGGApfApmG 687 G144-30UGUAAACAmUmCmCmUmGmCmGmAmCmAmGGApfApmG 688 G145-30UGUAAACAmUmCmCmUmGmCmGmAmCmAGGApfApmG 689 G146-30UGUAAACAmUmCmCmUmGmCmGmAmCAGGApfApmG 690 G147-30UGUAAACAUCmCmUmCmGmAmCmAmGGApfApmG 691 G148-30UGUAAACAUCmCmUmCmGmAmCmAGGApfApmG 692 G149-30UGUAAACAUCmCmUmCmGmAmCAGGApfApmG 693 G150-30UGUAAACAUmCmCmUmCmGmAmCmAmGGApfApmG 694 G151-30UGUAAACAUmCmCmUmCmGmAmCmAGGApfApmG 695 G152-30UGUAAACAUmCmCmUmCmGmAmCAGGApfApmG 696 G153-30UGUAAACAmUmCmCmUmCmGmAmCmAmGGApfApmG 697 G154-30UGUAAACAmUmCmCmUmCmGmAmCmAGGApfApmG 698 G155-30UGUAAACAmUmCmCmUmCmGmAmCAGGApfApmG 699 G156-30UGUAAACAUCmCmUmGmCmGmAmCmUmCAGpfCpmU 700 G157-30UGUAAACAUCmCmUmGmCmGmAmCmUCAGpfCpmU 701 G158-30UGUAAACAUCmCmUmGmCmGmAmCUCAGpfCpmU 702 G159-30UGUAAACAUmCmCmUmGmCmGmAmCmUmCAGpfCpmU 703 G160-30UGUAAACAUmCmCmUmGmCmGmAmCmUCAGpfCpmU 704 G161-30UGUAAACAUmCmCmUmGmCmGmAmCUCAGpfCpmU 705 G162-30UGUAAACAmUmCmCmUmGmCmGmAmCmUmCAGpfCpmU 706 G163-30UGUAAACAmUmCmCmUmGmCmGmAmCmUCAGpfCpmU 707 G164-30UGUAAACAmUmCmCmUmGmCmGmAmCUCAGpfCpmU 708 G165-30UGUAAACAUCmCmCmUmCmAmCmUmCmUCApfGpmC 709 G166-30UGUAAACAUCmCmCmUmCmAmCmUmCUCApfGpmC 710 G167-30UGUAAACAUCmCmCmUmCmAmCmUCUCApfGpmC 711 G168-30UGUAAACAUmCmCmCmUmCmAmCmUmCmUCApfGpmC 712 G169-30UGUAAACAUmCmCmCmUmCmAmCmUmCUCApfGpmC 713 G170-30UGUAAACAUmCmCmCmUmCmAmCmUCUCApfGpmC 714 G171-30UGUAAACAmUmCmCmCmUmCmAmCmUmCmUCApfGpmC 715 G172-30UGUAAACAmUmCmCmCmUmCmAmCmUmCUCApfGpmC 716 G173-30UGUAAACAmUmCmCmCmUmCmAmCmUCUCApfGpmC 717 G174-30UGUAAACAUCmCmCmUmCmAmCmUmCAGpfCpmU 718 G175-30UGUAAACAUCmCmCmUmCmAmCmUCAGpfCpmU 719 G176-30UGUAAACAUCmCmCmUmCmAmCUCAGpfCpmU 720 G177-30UGUAAACAUmCmCmCmUmCmAmCmUmCAGpfCpmU 721 G178-30UGUAAACAUmCmCmCmUmCmAmCmUCAGpfCpmU 722 G179-30UGUAAACAUmCmCmCmUmCmAmCUCAGpfCpmU 723 G180-30UGUAAACAmUmCmCmCmUmCmAmCmUmCAGpfCpmU 724 G181-30UGUAAACAmUmCmCmCmUmCmAmCmUCAGpfCpmU 725 G182-30UGUAAACAmUmCmCmCmUmCmAmCUCAGpfCpmU 726 G183-30UGUAAACAUCmCmCmAmCmAmCmUmCmUCApfGpmC 727 G184-30UGUAAACAUCmCmCmAmCmAmCmUmCUCApfGpmC 728 G185-30UGUAAACAUCmCmCmAmCmAmCmUCUCApfGpmC 729 G186-30UGUAAACAUmCmCmCmAmCmAmCmUmCmUCApfGpmC 730 G187-30UGUAAACAUmCmCmCmAmCmAmCmUmCUCApfGpmC 731 G188-30UGUAAACAUmCmCmCmAmCmAmCmUCUCApfGpmC 732 G189-30UGUAAACAmUmCmCmCmAmCmAmCmUmCmUCApfGpmC 733 G190-30UGUAAACAmUmCmCmCmAmCmAmCmUmCUCApfGpmC 734 G191-30UGUAAACAmUmCmCmCmAmCmAmCmUCUCApfGpmC 735 G192-30UGUAAACAUCmCmCmAmCmAmCmUmCAGpfCpmU 736 G193-30UGUAAACAUCmCmCmAmCmAmCmUCAGpfCpmU 737 G194-30UGUAAACAUCmCmCmAmCmAmCUCAGpfCpmU 738 G195-30UGUAAACAUmCmCmCmAmCmAmCmUmCAGpfCpmU 739 G196-30UGUAAACAUmCmCmCmAmCmAmCmUCAGpfCpmU 740 G197-30UGUAAACAUmCmCmCmAmCmAmCUCAGpfCpmU 741 G198-30UGUAAACAmUmCmCmCmAmCmAmCmUmCAGpfCpmU 742 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G177-205UCCUUCAUUmCmCmAmUmCmGmGmAmGUGCsfUsmG 421 G178-205UCCUUCAUUmCmCmAmUmCmGmGmAGUGCsfUsmG 422 G179-205UCCUUCAUmUmCmCmAmUmCmGmGmAmGmUGCsfUsmG 423 G180-205UCCUUCAUmUmCmCmAmUmCmGmGmAmGUGCsfUsmG 424 G181-205UCCUUCAUmUmCmCmAmUmCmGmGmAGUGCsfUsmG 425 G182-205UCCUUCAUUCmCmUmCmCmGmGmAmGUCsfUsmG 426 G183-205UCCUUCAUUCmCmUmCmCmGmGmAGUCsfUsmG 427 G184-205UCCUUCAUUCmCmUmCmCmGmGAGUCsfUsm? 428 G185-205UCCUUCAUUmCmCmUmCmCmGmGmAmGUCsfUsmG 429 G186-205UCCUUCAUUmCmCmUmCmCmGmGmAGUCsfUsmG 430 G187-205UCCUUCAUUmCmCmUmCmCmGmGAGUCsfUsmG 431 G188-205UCCUUCAUmUmCmCmUmCmCmGmGmAmGUCsfUsmG 432 G189-205UCCUUCAUmUmCmCmUmCmCmGmGmAGUCsfUsmG 433 G190-205UCCUUCAUmUmCmCmUmCmCmGmGAGUCsfUsmG 434 G191-205UCCUUCAUUCmCmUmCmCmGmGmAmGUGsfUsmG 435 G192-205UCCUUCAUUCmCmUmCmCmGmGmAGUGsfUsmG 436 G193-205UCCUUCAUUCmCmUmCmCmGmGAGUGsfUsmG 437 G194-205UCCUUCAUUmCmCmUmCmCmGmGmAmGUGsfUsmG 438 G195-205UCCUUCAUUmCmCmUmCmCmGmGmAGUGsfUsmG 439 G196-205UCCUUCAUUmCmCmUmCmCmGmGAGUGsfUsmG 440 G197-205UCCUUCAUmUmCmCmUmCmCmGmGmAmGUGsfUsmG 441 G198-205UCCUUCAUmUmCmCmUmCmCmGmGmAGUGsfUsmG 442 G199-205UCCUUCAUmUmCmCmUmCmCmGmGAGUGsfUsmG 443 G206-205UCCUUCAUACmCmAmCmCmGmGmAmGUCpfUpmG 846 G207-205UCCUUCAUACmCmAmCmCmGmGmAGUCpfUpmG 847 G208-205UCCUUCAUACmCmAmCmCmGmGAGUCpfUpmG 848 G209-205UCCUUCAUAmCmCmAmCmCmGmGmAmGUCpfUpmG 849 G210-205UCCUUCAUAmCmCmAmCmCmGmGmAGUCpfUpmG 850 G211-205UCCUUCAUAmCmCmAmCmCmGmGAGUCpfUpmG 851 G212-205UCCUUCAUmAmCmCmAmCmCmGmGmAmGUCpfUpmG 852 G213-205UCCUUCAUmAmCmCmAmCmCmGmGmAGUCpfUpmG 853 G214-205UCCUUCAUmAmCmCmAmCmCmGmGAGUCpfUpmG 854 G215-205UCCUUCAUCCmCmAmCmCmGmGmAmGUCpfUpmG 855 G216-205UCCUUCAUCCmCmAmCmCmGmGmAGUCpfUpmG 856 G217-205UCCUUCAUCCmCmAmCmCmGmGAGUCpfUpmG 857 G218-205UCCUUCAUCmCmCmAmCmCmGmGmAmGUCpfUpmG 858 G219-205UCCUUCAUCmCmCmAmCmCmGmGmAGUCpfUpmG 859 G220-205UCCUUCAUCmCmCmAmCmCmGmGAGUCpfUpmG 860 G221-205UCCUUCAUmCmCmCmAmCmCmGmGmAmGUCpfUpmG 861 G222-205UCCUUCAUmCmCmCmAmCmCmGmGmAGUCpfUpmG 862 G223-205UCCUUCAUmCmCmCmAmCmCmGmGAGUCpfUpmG 863 G224-205UCCUUCAUAUmCmCmAmCmCmGmGmAmGUCpfUpmG 864 G225-205UCCUUCAUAUmCmCmAmCmCmGmGmAGUCpfUpmG 865 G226-205UCCUUCAUAUmCmCmAmCmCmGmGAGUCpfUpmG 866 G227-205UCCUUCAUAmUmCmCmAmCmCmGmGmAmGUCpfUpmG 867 G228-205UCCUUCAUAmUmCmCmAmCmCmGmGmAGUCpfUpmG 868 G229-205UCCUUCAUAmUmCmCmAmCmCmGmGAGUCpfUpmG 869 G230-205UCCUUCAUmAmUmCmCmAmCmCmGmGmAmGUCpfUpmG 870 G231-205UCCUUCAUmAmUmCmCmAmCmCmGmGmAGUCpfUpmG 871 G232-205UCCUUCAUmAmUmCmCmAmCmCmGmGAGUCpfUpmG 872 G233-205UCCUUCAUCUmCmCmAmCmCmGmGmAmGUCpfUpmG 873 G234-205UCCUUCAUCUmCmCmAmCmCmGmGmAGUCpfUpmG 874 G235-205UCCUUCAUCUmCmCmAmCmCmGmGAGUCpfUpmG 875 G236-205UCCUUCAUCmUmCmCmAmCmCmGmGmAmGUCpfUpmG 876 G237-205UCCUUCAUCmUmCmCmAmCmCmGmGmAGUCpfUpmG 877 G238-205UCCUUCAUCmUmCmCmAmCmCmGmGAGUCpfUpmG 878 G239-205UCCUUCAUmCmUmCmCmAmCmCmGmGmAmGUCpfUpmG 879 G240-205UCCUUCAUmCmUmCmCmAmCmCmGmGmAGUCpfUpmG 880 G241-205UCCUUCAUmCmUmCmCmAmCmCmGmGAGUCpfUpmG 881 G242-205UCCUUCAUCAmCmCmAmCmCmGmGmAmGUCpfUpmG 882 G243-205UCCUUCAUCAmCmCmAmCmCmGmGmAGUCpfUpmG 883 G244-205UCCUUCAUCAmCmCmAmCmCmGmGAGUCpfUpmG 884 G245-205UCCUUCAUCmAmCmCmAmCmCmGmGmAmGUCpfUpmG 885 G246-205UCCUUCAUCmAmCmCmAmCmCmGmGmAGUCpfUpmG 886 G247-205UCCUUCAUCmAmCmCmAmCmCmGmGAGUCpfUpmG 887 G248-205UCCUUCAUmCmAmCmCmAmCmCmGmGmAmGUCpfUpmG 888 G249-205UCCUUCAUmCmAmCmCmAmCmCmGmGmAGUCpfUpmG 889 G250-205UCCUUCAUmCmAmCmCmAmCmCmGmGAGUCpfUpmG 890 G251-205UCCUUCAUAUmCmCmUmCmCmGmGmAmGUCpfUpmG 891 G252-205UCCUUCAUAUmCmCmUmCmCmGmGmAGUCpfUpmG 892 G253-205UCCUUCAUAUmCmCmUmCmCmGmGAGUCpfUpmG 893 G254-205UCCUUCAUAmUmCmCmUmCmCmGmGmAmGUCpfUpmG 894 G255-205UCCUUCAUAmUmCmCmUmCmCmGmGmAGUCpfUpmG 895 G256-205UCCUUCAUAmUmCmCmUmCmCmGmGAGUCpfUpmG 896 G257-205UCCUUCAUmAmUmCmCmUmCmCmGmGmAmGUCpfUpmG 897 G258-205UCCUUCAUmAmUmCmCmUmCmCmGmGmAGUCpfUpmG 898 G259-205UCCUUCAUmAmUmCmCmUmCmCmGmGAGUCpfUpmG 899 P001-205Amino C6-mGmAmUUUCAGUGGAGUGAAGmUmUmC 444 G001-338UCCAGCAUCAmGmUmGmAmUmUmUmUGUsfUsmG 445 G002-338UCCAGCAUCAmGmUmGmAmUmUmUUGUsfUsmG 446 G003-338UCCAGCAUCAmGmUmGmAmUmUUUGUsfUsmG 447 G004-338UCCAGCAUCmAmGmUmGmAmUmUmUmUGUsfUsmG 448 G005-338UCCAGCAUCmAmGmUmGmAmUmUmUUGUsfUsmG 449 G006-338UCCAGCAUCmAmGmUmGmAmUmUUUGUsfUsmG 450 G007-338UCCAGCAUmCmAmGmUmGmAmUmUmUmUGUsfUsmG 451 G008-338UCCAGCAUmCmAmGmUmGmAmUmUmUUGUsfUsmG 452 G009-338UCCAGCAUmCmAmGmUmGmAmUmUUUGUsfUsmG 453 P001-338Amino C6-mAmAmCAAUAUCCUGGUGCUGAmGmUmG 454 G001-375UUUGUUCGUUmCmGmGmCmUmCmGmCGUsfGsmA 455 G002-375UUUGUUCGUUmCmGmGmCmUmCmGCGUsfGsmA 456 G003-375UUUGUUCGUUmCmGmGmCmUmCGCGUsfGsmA 457 G004-375UUUGUUCGUmUmCmGmGmCmUmCmGmCGUsfGsmA 458 G005-375UUUGUUCGUmUmCmGmGmCmUmCmGCGUsfGsmA 459 G006-375UUUGUUCGUmUmCmGmGmCmUmCGCGUsfGsmA 460 G007-375UUUGUUCGmUmUmCmGmGmCmUmCmGmCGUsfGsmA 461 G008-375UUUGUUCGmUmUmCmGmGmCmUmCmGCGUsfGsmA 462 G009-375UUUGUUCGmUmUmCmGmGmCmUmCGCGUsfGsmA 463 P001-375Amino C6-mGmCmGACGAGCCCCUCGCACAAmAmCmC 464 G001-E1 UUGAGAAGGAGGCUGCUGAGA465 P001-E1 UCAGCAGCCUCCUUCUAA 466

TABLE 4 Mimic/mimetic composition Mimic name Guide Strand PassengerStrand M30-021 G039-30 (SEQ ID NO. 67) P014-30 (SEQ ID NO. 95) M30-025G032-30 (SEQ ID NO. 65) P014-30 (SEQ ID NO. 95) M30-033 G042-30 (SEQ IDNO. 69) P014-30 (SEQ ID NO. 95) M30-034 G042-30 (SEQ ID NO. 69) P123-30(SEQ ID NO. 96) M30-035 G121-30 (SEQ ID NO. 83) P123-30 (SEQ ID NO. 96)M30-036 G122-30 (SEQ ID NO. 84) P123-30 (SEQ ID NO. 96) M30-037 G011-30(SEQ ID NO. 55) P123-30 (SEQ ID NO. 96) M30-038 G121-30 (SEQ ID NO. 83)P014-30 (SEO ID NO. 95) M30-039 G039-30 (SEQ ID NO. 67) P123-30 (SEQ IDNO. 96) M30-040 G032-30 (SEQ ID NO. 65) P123-30 (SEQ ID NO. 96) M30-042G128-30 (SEQ ID NO. 85) P123-30 (SEQ ID NO. 96) M30-043 G129-30 (SEQ IDNO. 86) P123-30 (SEQ ID NO. 96) M30-044 G032-30 (SEQ ID NO. 65) P126-30(SEQ ID NO. 97) M30-046 G130-30 (SEQ ID NO. 87) P123-30 (SEQ ID NO. 96)M30-047 G130-30 (SEQ ID NO. 87) P126-30 (SEQ ID NO. 97) M30-048 G132-30(SEQ ID NO. 88) P123-30 (SEQ ID NO. 96) M30-049 G133-30 (SEQ ID NO. 89)P131-30 (SEQ ID NO. 99) M29-002 miR-29a-3p (SEQ ID NO. 12) P009-29 (SEQID NO. 175) M29-004 miR-29b-3p (SEQ ID NO. 13) P004-29 (SEQ ID NO. 170)M29-005 miR-29b-3p (SEQ ID NO. 13) P005-29 (SEQ ID NO. 171) M29-006miR-29b-3p (SEQ ID NO. 13) P006-29 (SEQ ID NO. 172) M29-007 miR-29b-3p(SEQ ID NO. 13) P007-29 (SEQ ID NO. 173) M29-008 miR-29b-3p (SEQ ID NO.13) P011-29 (SEQ ID NO. 176) M29-009 G013-29 (SEQ ID NO. 108) P007-29(SEQ ID NO. 173) M29-010 G013-29 (SEQ ID NO. 108) P006-29 (SEQ ID NO.172) M29-011 G003-29 (SEQ ID NO. 102) P007-29 (SEQ ID NO. 173) M29-012G003-29 (SEQ ID NO. 102) P011-29 (SEQ ID NO. 176) M29-013 G004-29 (SEQID NO. 103) P011-29 (SEQ ID NO. 176) M29-014 G004-29 (SEQ ID NO. 103)P007-29 (SEQ ID NO. 173) M29-015 G005-29 (SEQ ID NO. 104) P011-29 (SEQID NO. 176) M29-016 G005-29 (SEQ ID NO. 104) P007-29 (SEQ ID NO. 173)M29-017 G006-29 (SEQ ID NO. 105) P011-29 (SEQ ID NO. 176) M29-018G006-29 (SEQ ID NO. 105) P007-29 (SEQ ID NO. 173) M29-019 G007-29 (SEQID NO. 106) P011-29 (SEQ ID NO. 176) M29-020 G007-29 (SEQ ID NO. 106)P007-29 (SEQ ID NO. 173) M29-023 miR-29a-3p (SEQ ID NO. 12) P008-29 (SEQID NO. 174) M29-024 G009-29 (SEQ ID NO. 127) P007-29 (SEQ ID NO. 173)M29-025 G009-29 (SEQ ID NO. 127) P011-29 (SEQ ID NO. 176) M29-026G009-29 (SEQ ID NO. 127) P005-29 (SEQ ID NO. 171) M29-027 G010-29 (SEQID NO. 128) P007-29 (SEQ ID NO. 173) M29-028 G010-29 (SEQ ID NO. 128)P011-29 (SEQ ID NO. 176) M29-029 G011-29 (SEQ ID NO. 129) P007-29 (SEQID NO. 173) M29-030 G011-29 (SEQ ID NO. 129) P011-29 (SEQ ID NO. 176)M29-031 G012-29 (SEQ ID NO. 130) P007-29 (SEQ ID NO. 173) M29-032G012-29 (SEQ ID NO. 130) P011-29 (SEQ ID NO. 176) M29-033 G014-29 (SEQID NO. 131) P007-29 (SEQ ID NO. 173) M29-034 G014-29 (SEQ ID NO. 131)P011-29 (SEQ ID NO. 176) M29-035 G015-29 (SEQ ID NO. 620) P011-29 (SEQID NO. 176) M29-036 G015-29 (SEQ ID NO. 620) P012-29 (SEQ ID NO. 838)M29-037 G016-29 (SEQ ID NO. 835) P011-29 (SEQ ID NO. 176) M29-038G016-29 (SEQ ID NO. 835) P012-29 (SEQ ID NO. 838) M29-039 G007-29 (SEQID NO. 106) P013-29 (SEQ ID NO. 177) M29-040 G017-29 (SEQ ID NO. 836)P013-29 (SEQ ID NO. 177) M29-041 G018-29 (SEQ ID NO. 837) P014-29 (SEQID NO. 839) E1-001 G001-E1 (SEQ ID NO. 465) P001-E1 (SEQ ID NO. 466)

For example, an engineered oligonucleotide or salt thereof can compriseat least about 80% sequence identity to an oligonucleotide of any one ofSEQ ID NOs: 1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89,100-154, 184-201, 205-222, 225-233, 235-243, 245-443, 445-453, 455-463,465, 620, 624-825, 835-837, 840-899, and 901-949. For example, anengineered oligonucleotide or salt thereof can comprise at least about90% sequence identity to an oligonucleotide of any one of SEQ ID NOs:1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89, 100-154,184-201, 205-222, 225-233, 235-243, 245-443, 445-453, 455-463, 465, 620,624-825, 835-837, 840-899, and 901-949. In some cases, an engineeredoligonucleotide or salt thereof can comprise from about 80% to 100%sequence identity to an oligonucleotide of any one of SEQ ID NOs: 1-5,12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89, 100-154, 184-201,205-222, 225-233, 235-243, 245-443, 445-453, 455-463, 465, 620, 624-825,835-837, 840-899, and 901-949. In some cases, an engineeredoligonucleotide or salt thereof can comprise from about 85% to 100%sequence identity to an oligonucleotide of any one of SEQ ID NOs: 1-5,12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89, 100-154, 184-201,205-222, 225-233, 235-243, 245-443, 445-453, 455-463, 465, 620, 624-825,835-837, 840-899, and 901-949. In some cases, an engineeredoligonucleotide or salt thereof can comprise at least 80% sequenceidentity to at least about 10 contiguous bases of any one of SEQ ID NOs:1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89, 100-154,184-201, 205-222, 225-233, 235-243, 245-443, 445-453, 455-463, 465, 620,624-825, 835-837, 840-899, and 901-949. In some cases, an engineeredoligonucleotide or salt thereof can comprise at least 85% sequenceidentity to at least about 10 contiguous bases of any one of SEQ ID NOs:1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 52-89, 100-154,184-201, 205-222, 225-233, 235-243, 245-443, 445-453, 455-463, 465, 620,624-825, 835-837, 840-899, and 901-949.

In some cases, the engineered oligonucleotide can comprise at leastabout: 70%, 75%, 80%, 85%, 90%, 95% sequence identity to any one of SEQID NOs: 1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45, 100-126,245-263, 465, 624-643, 840-845, and 901-949, or any combination thereof.

In some cases, a second strand can comprise at least about: 70%, 75%,80%, 85%, 90%, 95% sequence identity to any one of SEQ ID NOs: 6-11,15-18, 21-23, 26-27, 29, 31, 33, 35, 37, 46-51, 90-99, 155-183, 202-204,223-224, 234-244, 444, 454, 464, 466, 838-839, or any combinationthereof. In some cases, the second strand can comprise at least about:70%, 75%, 80%, 85%, 90%, 95% sequence identity to any one of: SEQ IDNOs: 51, 95-99, or any combination thereof.

For example, an engineered oligonucleotide or salt thereof can compriseat least about 80% sequence identity to an oligonucleotide of any one ofSEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 500, SEQ IDNO: 513, SEQ ID NO: 518, SEQ ID NO: 476, SEQ ID NO: 481, or SEQ ID NO:495. For example, an engineered oligonucleotide or salt thereof cancomprise at least about 90% sequence identity to an oligonucleotide ofany one of SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO:500, SEQ ID NO: 513, SEQ ID NO: 518, SEQ ID NO: 476, SEQ ID NO: 481, orSEQ ID NO: 495. In some cases, an engineered oligonucleotide or saltthereof can comprise from about 80% to 100% sequence identity to anoligonucleotide of any one of SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO:589, SEQ ID NO: 500, SEQ ID NO: 513, SEQ ID NO: 518, SEQ ID NO: 476, SEQID NO: 481, or SEQ ID NO: 495. In some cases, an engineeredoligonucleotide or salt thereof can comprise from about 85% to 100%sequence identity to an oligonucleotide of any one of SEQ ID NO: 587,SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 500, SEQ ID NO: 513, SEQ IDNO: 518, SEQ ID NO: 476, SEQ ID NO: 481, or SEQ ID NO: 495. In somecases, an engineered oligonucleotide or salt thereof can comprise atleast 80% sequence identity to at least about 10 contiguous bases of anyone of SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 500,SEQ ID NO: 513, SEQ ID NO: 518, SEQ ID NO: 476, SEQ ID NO: 481, or SEQID NO: 495. In some cases, an engineered oligonucleotide or salt thereofcan comprise at least 85% sequence identity to at least about 10contiguous bases of any one of SEQ ID NO: 587, SEQ ID NO: 588, SEQ IDNO: 589, SEQ ID NO: 500, SEQ ID NO: 513, SEQ ID NO: 518, SEQ ID NO: 476,SEQ ID NO: 481, or SEQ ID NO: 495.

In some cases, an engineered oligonucleotide or salt thereof cancomprise at least about: 1%, 2%, 5% 7%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, or 20% lower Gibbs free energy (ΔG) of bindingto the RNA sequence at about 37 degrees Celsius and at about pH 7.2. Insome cases, an engineered oligonucleotide or salt thereof can bind theRNA sequence at about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 degrees Celsius. In somecases, an engineered oligonucleotide or salt thereof can bind the RNAsequence at a pH of about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, or 7.8.

The terms “administer,” “administering”, “administration,” and the like,as used herein, can refer to methods that can be used to enable deliveryof compounds or compositions to the desired site of biological action.Delivery can include direct application to the affect tissue or regionof the body. Delivery can include a parenchymal injection, anintra-thecal injection, an intra-ventricular injection, or anintra-cisternal injection. A composition provided herein can beadministered by any method. A method of administration can be byinhalation, intraarterial injection, intracerebroventricular injection,intracisternal injection, intramuscular injection, intraorbitalinjection, intraparenchymal injection, intraperitoneal injection,intraspinal injection, intrathecal injection, intravenous injection,intraventricular injection, stereotactic injection, subcutaneousinjection, or any combination thereof. Delivery can include parenteraladministration (including intravenous, subcutaneous, intrathecal,intraperitoneal, intramuscular, intravascular or infusion), oraladministration, inhalation administration, intraduodenal administration,rectal administration. Delivery can include topical administration (suchas a lotion, a cream, an ointment) to an external surface of a surface,such as a skin. In some instances, a subject can administer thecomposition in the absence of supervision. In some instances, a subjectcan administer the composition under the supervision of a medicalprofessional (e.g., a physician, nurse, physician's assistant, orderly,hospice worker, etc.). In some cases, a medical professional canadminister the composition. In some cases, a cosmetic professional canadminister the composition.

The term “subject,” “host,” “individual,” and “patient” are as usedinterchangeably here-in to refer to animals, typically mammaliananimals. Any suitable mammal can be administered a composition asdescribed herein (such as an engineered oligonucleotide) or treated by amethod as described herein. Non-limiting examples of mammals includehumans, non-human primates (e.g., apes, gibbons, chimpanzees,orangutans, monkeys, macaques, and the like), domestic animals (e.g.,dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs)and experimental animals (e.g., mouse, rat, rabbit, guinea pig). Mammalscan be any age or at any stage of development, for example a mammal canbe neonatal, infant, adolescent, adult or in utero. In some embodimentsa mammal can be a human. Humans can be more than about: 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, or 100 years of age. Humans can be less thanabout: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 years of age.A mammal can be male or female. In some embodiments a subject can be ahuman. In some embodiments, a subject may be suspected of having adisease or condition. The subject can be a patient, such as a patientbeing treated for a condition or a disease, such as a cancer, a fibrosiscondition, or a viral infection patient. The subject may be predisposedto a risk of developing a condition or a disease such as cancer. Thesubject may be in remission from a condition or a disease, such as acancer patient. The subject may be healthy.

The term “mammalian cell” can refer to any mammalian cell, typically ahuman cell. In some embodiments the human cell can be a cell of head orneck tissue, a skin cell, a cervical cell, a prostate cell, a stem cell,a bone cell, a blood cell, a muscle cell, a fat cell, a nerve cell, anendothelial cell, sperm cell, egg cell, cancer cell, barrier cell,hormone-secreting cell, exocrine-secretory cell, epithelial cell, oralcell, sensory transducer cell, autonomic neuron cell, peripheral neuroncell, central nervous neuron cell, secretory cell, barrier cell, musclecell, cardiac muscle cell, white blood cell, germ cell, nurse cell,kidney cell, or any combination thereof.

As used herein, cancer refers to a disease caused by an uncontrolleddivision of abnormal cells in a part of the body. Those cells can be ofdifferent types selected from a list maintained by the National CancerInstitute (https://www.cancer.gov/types).

As used herein, “treating” of cancer can include one or more of:reducing the frequency and/or severity of symptoms, elimination ofsymptoms and/or their underlying cause, and improvement or remediationof damage. For example, treatment of cancer can include, for example,relieving the pain experienced by a mammal suffering from cancer, and/orcausing the regression or disappearance of cancer. Treating may alsoinclude: reduced malaise, cessation of cancer side effects, abatement oftumors, or any combination thereof. Treating can include administeringan engineered oligonucleotide of SEQ ID NOs: 1-5, 12-14, 19-20, 24-25,28, 30, 32, 34, 36, 38-45, 52-89, 100-154, 184-201, 205-222, 225-233,235-243, 245-443, 445-453, 455-463, 465, 620, 624-825, 835-837, and840-899, or any combination thereof. As used herein, “treating” offibrosis, scarring, or both can include one or more of: reducing thefrequency and/or severity of symptoms, elimination of symptoms and/ortheir underlying cause, and improvement or remediation of damage. Forexample, treatment of fibrosis can include, for example, relievingshortness of breath experienced by a mammal suffering from pulmonaryfibrosis, and/or causing the regression or disappearance of fibrosis.Treating may also include: reduced malaise, cessation of fibrosis orscarring side effects, abatement of fibrotic tissue, or any combinationthereof. Treating can include administering an engineeredoligonucleotide of SEQ ID NOs: 1-5, 12-14, 19-20, 38-45, 52-89, 100-154,184-201, 620, 624-825, and 835-837, or any combination thereof. As usedherein, “treating” of a viral infection can include one or more of:reducing the frequency and/or severity of symptoms, elimination ofsymptoms and/or their underlying cause, elimination of the infectiousdisease, and improvement or remediation of damage. For example,treatment of a viral infection can include, for example, relieving thecough experienced by a mammal suffering from a viral infection, and/orcausing the regression or disappearance of a viral infection. Treatingmay also include: reduced malaise, cessation of viral infection sideeffects, abatement of a viral infection, or any combination thereof.Treating can include administering an engineered oligonucleotide of SEQID NOs: 1-5, 12-14, 19-20, 38-45, 52-89, 100-154, 184-201, 620, 624-825,and 835-837, or any combination thereof.

As used herein, “treating” of muscular dystrophy can include one or moreof: reducing the frequency and/or severity of symptoms, elimination ofsymptoms and/or their underlying cause, and improvement or remediationof damage. For example, treatment of muscular dystrophy can include, forexample, relieving the muscle weakness experienced by a mammal sufferingfrom muscular dystrophy, and/or causing the regression or disappearanceof muscle weakness. Treating may also include: reduced malaise,cessation of myotonia, abatement of muscle pain, or any combinationthereof. Treating can include administering an engineeredoligonucleotide of SEQ ID NOs: 12-14, 30, 100-154, 235-243, 620,835-837, and 901-949, or any combination thereof.

TABLE 5 ASOs SEQ ID Targeted mRNA SEQ Oligo Sequence (5′-3′) NO: ID NOS:AS-DX- TCCAAACGAGTCTCCG 901 954, 1017, 1046, 1112 003 AS-DX-GATTCTGAAACCAGA 902 958, 994, 1004, 1040, 004 1057, 1111 AS-DX-GCGGGCGCCCTGCCAC 903 1030, 1047, 1104 005 AS-DX- TCATCCAGCAGCAGGC 904981, 1025, 1068, 1069, 006 1113 AS-DX- TAGCCAGCCAGGTGTT 905966, 967, 997, 1056 007 AS-DX- CAGCGTCGGAAGGTGG 906955, 999, 1071, 1092, 008 1118 AS-DX- TAGACAGCGTCGGAAG 9071021, 1051, 1094 009 AS-DX- ATAGGATCCACAGGGA 908 1032, 1065, 1107 010AS-DX- TCTATAGGATCCACAG 909 1032, 1067, 1075, 1106 011 AS-DX-GCACTAATCATCCAGG 910 957, 1035, 1042 012 AS-DX- CAGCGTCGGAAGGTG 911955, 999, 1071, 1092, 014 1118 AS-DX- CCTAGACAGCGTCGGAAGGT 912955, 999, 1071, 1092, 015 1118 AS-DX- ATAGGATCCACAGGGAGG 913 1012, 1065018 AS-DX- CGGCTCTGGGATCCCCGG 914 973, 1011 019 AS-DX- GGGGCGGAGACACGCCC915 986, 1028 021 AS-DX- AGAAGGCAGGAATCCCAG 916 976, 1019 022 AS-DX-GCAGGAATCCCAGGCCGG 917 972, 1010 023 AS-DX- GGAGTCTCTCACCGGGCC 918984, 1026, 1076 025 AS-DX- GCGAGGCGGCCTCTTCCG 919 964, 989, 1033, 1064036 AS-DX- GCCTCCAGCTCCCCCGGG 920 980, 992, 1024 037 AS-DX-GGTGTCGGGAGGGCCAT 921 1006, 1039, 1108 038 AS-DX- CGGTATTCTTCCTCGCTG 922952, 963, 991, 1036, 1082 055 AS-DX- GGGCATTTTAATATATCTCT 9231003, 1074, 1085 060 GAACT AS-DX- TATCTTCTGAACTAATCATC 924957, 1035, 1042 061 CA AS-DX- CAGGAGATGTAACTCTAATC 9251018, 1059, 1090, 1101, 062 CAG 1105, 1119 AS-DX- CTCTCACCGGGCCTAGACCT926 987, 1034, 1077, 1109 063 AGAAG AS-DX- TGCGCACTGCGCGCAGGTCT 9271014, 1043, 1091, 1103 064 AGCCA AS-DX- ACTGCGCGCAGGTCTAGCCA 9281031, 1001, 1072, 1084, 065 GGAAG 1089 AS-DX- CGGGGTGCGCACTGCGCGCA 929998, 1048, 1049, 1050, 066 GGTCT 1097, 1102 AS-DX- TGCGCACTGCGCGCAGGTCT930 960, 978, 1001 067 AGCCAGGAAG AS-DX- ACTGCGCGCAGGTCTAGCCA 931974, 1031, 1089, 1110 068 GGAAGCGGGC AS-DX- ACCCGACCCCGTCCCAACCC 932968, 1000, 1081, 1095 069 CGCGT AS-DX- TGGGCTGGTGGAGAGGCAG 933975, 1013, 1054, 1120 070 AS-DX- TTCCCTGCATGTTTCCGGGT 934 983, 1061 074GCCCG AS-DX- CTTCCCTGCATGTTTCCGG 935 971, 1063 075 AS-DX-TGTGGCTCTCGTTCATTTC 936 977, 1096 076 AS-DX- CTCCGTGGGAGTCTTGAGT 937950, 961, 982, 1070, 1073 077 GTGCCA AS-DX- TGGAACTGAACCTCCGTGG 938965, 988, 1066 078 AS-DX- CACCCCTTCATGAATGGCGCC 939 979, 995, 1052, 1083080 AS-DX- ACAGGCTCCACCCCTTCATG 940 970, 1044, 1045, 1088, 081 1114AS-DX- TTCCGCTCAAAGCAGGCCTC 941 953, 1022, 1116 082 AS-DX-AAAGCGATCCTTCTCAAAGGC 942 993, 1023, 1041, 1078 083 TCGG AS-DX-CCTGCGCGGGCGCCCTGCCGC 943 951, 1030, 1098, 1115 084 AS-DX-TATCTCTGAACTAATCATC 944 956, 1005, 1053 085 AS-DX-AGCGCCTGGCGGCGGAACGCAG 945 990, 1058, 1093 086 ACC AS-DX-ATCTCTGCCCGCCTTCCCTCCC 946 985, 1087, 1100 087 GCC AS-DX-AAACCAGATCTGAATCCTGGAC 947 959, 962, 1029, 1055, 088 1060 AS-DX-TTTCTAGGAGAGGTTGCGCCTG 948 969, 1008, 1079 089 AS-DX-CCTAGACAGCGTCGGAAGGTAG 949 955, 999, 1071, 1092, 097 1118

A regulatory non-coding RNA (ncRNA) comprises short non-coding RNAsequences expressed in a genome that regulates expression or function ofother biomolecules in mammalian cells. An ncRNA is generally <200nucleotides in length and can be single stranded or double stranded, andmay form non-linear secondary or tertiary structures. An ncRNA cancomprise exogenously derived small interfering RNA (siRNA), MicroRNA(miRNA), small nuclear RNA (U-RNA), Small nucleolar RNA (snoRNA),Piwi-interacting RNA (piRNA), repeat associated small interfering RNA(rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA derived smallRNA (tsRNA), ribosomal RNA derived small RNA (rsRNA), large non-codingRNA derived small RNA (IncsRNA), or a messenger RNA derived small RNA(msRNA).

An engineered oligonucleotide can comprise DNA or RNA. In some cases, anengineered oligonucleotide can comprise a plurality of nucleotides. Insome cases, an engineered oligonucleotide can comprise an artificialnucleic acid analogue. In some cases, an engineered oligonucleotide cancomprise DNA, can comprise cell-free DNA, cDNA, fetal DNA, viral DNA, ormaternal DNA. In some cases, an engineered oligonucleotide can comprisean shRNA, or siRNA, an ncRNA mimic, a short-harpin RNA (shRNA), adicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), agapmer, a mixmer, double-stranded RNAs (dsRNA), single stranded RNAi,(ssRNAi), DNA-directed RNA interference (ddRNAi), an RNA activatingoligonucleotide (RNAa), or an exon skipping oligonucleotide. In somecases, an engineered oligonucleotide can comprise a completely syntheticmiRNA. A completely synthetic miRNA is one that is not derived or basedupon an ncRNA. Instead, a completely synthetic miRNA may be based uponan analysis of multiple potential target sequences or may be based uponisolated natural non-coding sequences that are not ncRNAs. One exampleof a completely synthetic miRNA is E1-001 (Table 4).

A diagnostic test can comprise an imaging procedure, a blood countanalysis, a tissue pathology analysis, a biomarker analysis, a biopsy, amagnetic resonance image procedure, a physical examination, a urinetest, an ultrasonography procedure, a genetic test, a liver functiontest, a positron emission tomography procedure, a X-ray, serology, anangiography procedure, an electrocardiography procedure, an endoscopy, adiagnostic polymerase chain reaction test (PCR), a pap smear, ahematocrit test, a skin allergy test, a urine test, a colonoscopy, anenzyme-linked immunosorbent assay (ELISA), microscopy analysis, bonemarrow examination, rapid diagnostic test, pregnancy test, organfunction test, toxicology test, infectious disease test, bodily fluidstest, or any combination thereof.

A pharmaceutical composition can comprise a first active ingredient. Thefirst active ingredient can comprise an engineered oligonucleotide asdescribed herein. The pharmaceutical composition can be formulated inunit dose form. The pharmaceutical composition can comprise apharmaceutically acceptable excipient, diluent, or carrier. Thepharmaceutical composition can comprise a second, third, or fourthactive ingredient, such as a second engineered oligonucleotide.

A composition described herein can compromise an excipient. An excipientcan comprise a pH agent (to minimize oxidation or degradation of acomponent of the composition), a stabilizing agent (to preventmodification or degradation of a component of the composition), abuffering agent (to enhance temperature stability), a solubilizing agent(to increase protein solubility), or any combination thereof. Anexcipient can comprise a surfactant, a sugar, an amino acid, anantioxidant, a salt, a non-ionic surfactant, a solubilizer, atriglyceride, an alcohol, or any combination thereof. An excipient cancomprise sodium carbonate, acetate, citrate, phosphate, poly-ethyleneglycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose,polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol,polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine,sodium citrate, trehalose, arginine, sodium acetate, acetate, HCl,disodium edetate, lecithin, glycerin, xanthan rubber, soy isoflavones,polysorbate 80, ethyl alcohol, water, teprenone, or any combinationthereof. An excipient can be an excipient described in the Handbook ofPharmaceutical Excipients, American Pharmaceutical Association (1986).

Administration or application of a composition disclosed herein can beperformed for a treatment duration of at least about at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100 days consecutive or nonconsecutive days.In some cases, a treatment duration can be from about 1 to about 30days, from about 2 to about 30 days, from about 3 to about 30 days, fromabout 4 to about 30 days, from about 5 to about 30 days, from about 6 toabout 30 days, from about 7 to about 30 days, from about 8 to about 30days, from about 9 to about 30 days, from about 10 to about 30 days,from about 11 to about 30 days, from about 12 to about 30 days, fromabout 13 to about 30 days, from about 14 to about 30 days, from about 15to about 30 days, from about 16 to about 30 days, from about 17 to about30 days, from about 18 to about 30 days, from about 19 to about 30 days,from about 20 to about 30 days, from about 21 to about 30 days, fromabout 22 to about 30 days, from about 23 to about 30 days, from about 24to about 30 days, from about 25 to about 30 days, from about 26 to about30 days, from about 27 to about 30 days, from about 28 to about 30 days,or from about 29 to about 30 days.

Administration or application of a composition disclosed herein can beperformed for a treatment duration of at least about 1 week, at leastabout 1 month, at least about 1 year, at least about 2 years, at leastabout 3 years, at least about 4 years, at least about 5 years, at leastabout 6 years, at least about 7 years, at least about 8 years, at leastabout 9 years, at least about 10 years, at least about 15 years, atleast about 20 years, or more. Administration can be performedrepeatedly over a lifetime of a subject, such as once a month or once ayear for the lifetime of a subject. Administration can be performedrepeatedly over a substantial portion of a subject's life, such as oncea month or once a year for at least about 1 year, 5 years, 10 years, 15years, 20 years, 25 years, 30 years, or more.

Administration or application of composition disclosed herein can beperformed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 times a day. In some cases,administration or application of composition disclosed herein can beperformed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 times a week. In some cases, administration orapplication of composition disclosed herein can be performed at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times amonth.

In some cases, a composition can be administered/applied as a singledose or as divided doses. In some cases, the compositions describedherein can be administered at a first time point and a second timepoint. In some cases, a composition can be administered such that afirst administration is administered before the other with a differencein administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours,16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 1 year or more.

In some cases, an engineered oligonucleotide or salt thereof comprisinga modification when contacted with an mRNA sequence can produce loweractivity of a polypeptide encoded by the mRNA sequence as compared tocontacting an equivalent amount of an otherwise comparableoligonucleotide that lacks the modification with the mRNA sequence. Insome cases, the lower activity can be at least about 1.2-fold lower. Insome cases, the lower activity can be at least about 1.5-fold lower. Insome cases, the lower activity can be at least about 1.7-fold lower. Insome cases, the lower activity can be at least about 2.0-fold lower. Insome cases, the lower activity can be about: 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5-fold lower. In some cases,the lower activity can be from about 1.2-fold to about 2.0-fold lower.In some cases, the lower activity can be from about 1.1-fold to about1.5-fold lower. In some cases, the lower activity can be from about1.1-fold to about 2.5-fold lower. In some cases, the lower activity canbe from about 1.2-fold to about 3.0-fold lower. In some cases, the loweractivity can be at least about 1.2-fold to about at least 10-fold lowerexpression. In some cases, the lower activity can be at least about14-fold lower. In some cases, the lower expression can be at least about18-fold lower expression. In some cases, the lower activity can beabout: 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20-fold lower. In some cases, the lower activity can be from about1.2-fold to about 14-fold. In some cases, the lower activity can be fromabout 1.1-fold to about 20-fold lower. In some cases, the lower activitycan be from about 1.2-fold to about 30-fold lower.

In some cases, an engineered oligonucleotide or salt thereof comprisinga modification when stored in a closed container placed in a room for atime period will remain at least about 80% of an initial amount of theengineered oligonucleotide or salt thereof. In some cases, theengineered oligonucleotide will remain at least about 70% the initialamount. In some cases, the engineered oligonucleotide will remain atleast about 90% the initial amount. In some cases, the engineeredoligonucleotide will remain at least about: 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%. In some cases, the engineered nucleotide can be atleast about 60% to about at least 80%. In some cases, the engineerednucleotide can be at least about 80% to at least about 99%. In somecases, the time period of storage can be at least 1 month. In somecases, the time period of storage can be at least about 3 months. Insome cases, the time period of storage can be at least about 1 year. Insome cases, the time period of storage can be at least about 1, 2, 4, 6,8, 12, 18, 24, 36, 48 or 60 months. In some cases, the time period ofstorage can be at least about 1 month to about at least 1 year. In somecases, the time period of storage can be at least about 6 months to atleast about 2 years. In some cases, the time period of storage can be atleast about 1 month to at least about 5 years.

The term “tissue” as used herein, can be any tissue sample. A tissue canbe a tissue suspected or confirmed of having a disease or condition. Atissue can be a sample that may be substantially healthy, substantiallybenign, or otherwise substantially free of a disease or a condition. Atissue can be a tissue removed from a subject, such as a tissue biopsy,a tissue resection, an aspirate (such as a fine needle aspirate), atissue washing, a cytology specimen, a bodily fluid, or any combinationthereof. A tissue can comprise cancerous cells, tumor cells,non-cancerous cells, or a combination thereof. A tissue can comprise ablood sample (such as a cell-free DNA sample). A tissue can be a samplethat may be genetically modified.In some cases, a disease or condition can comprise a viral infection, afibrosis condition, a cancer, a muscular dystrophy, or any combinationthereof. In some cases, the disease or condition can comprise the viralinfection. The viral infection can comprise a SARS-CoV infection, aSARS-CoV-2 infection, a MERS-CoV infection, a CoV-HKU1 infection, an HIVinfection, an HCV infection, or any combination thereof. The viralinfection can comprise a coronavirus infection. A coronavirus can be aSARS-CoV, a SARS-CoV-2, a CoV-HKU1, or a MERS-CoV. In some cases, theviral infection can comprise an HCV genotype 1 infection. In some cases,at least a portion of the RNA sequence can be encoded by a SARS-CoVgenome, a MERS-CoV genome, a CoV-HKU1 genome, an HIV genome, or anycombination thereof. In some cases, an engineered oligonucleotide can beselective for an RNA sequence. In some cases, an RNA sequence cancomprise at least about: 70%, 75%, 80%, 85%, 90%, 95% sequence identityto an RNA sequence of SEQ ID NOS: 500-531, 829-831 (Table 6), SEQ IDNOS: 474-499, 826-828 (Table 6), SEQ ID NOS: 532-554 (Table 6), SEQ IDNOS: 555-586 (Table 6), or any combination thereof. In some cases, asubject may suffer from a SARS-CoV-2 infection and the RNA sequence cancomprise at least about: 70%, 75%, 80%, 85%, 90%, 95% sequence identityto an RNA sequence of SEQ ID NOS: 500-531, 829-831 (Table 6). In somecases, a subject may suffer from a SARS-CoV infection and the RNAsequence can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%sequence identity to an RNA sequence of SEQ ID NOS: 474-499, 826-828(Table 6). In some cases, a subject may suffer from a MERS-CoV infectionand the RNA sequence can comprise at least about: 70%, 75%, 80%, 85%,90%, 95% sequence identity to an RNA sequence of SEQ ID NOS: 532-554(Table 6). In some cases, a subject may suffer from a CoV-HKU1 infectionand the RNA sequence can comprise at least about: 70%, 75%, 80a, 85,90%, 95 sequence identity to an RNA sequence of SEQ ID NOS: 555-586(Table 6). In some cases, at least a portion of the RNA sequence can beencoded in an HIV genome. In some cases, the RNA sequence can compriseat least about: 75%, 80%, 85%, 90%, 95% or more sequence identity to SEQID NO: 470, as determined by a BLAST pairwise sequence alignmentalgorithm. In some cases, the viral infection can be an HIV infection.In some cases, the RNA sequence can comprise at least about: 70%, 75%,80%, 85%, 90%, 95% or more sequence identity to SEQ ID NO: 471, SEQ IDNO: 472, or SEQ ID NO: 473, as determined by a BLAST pairwise sequencealignment algorithm.

TABLE 6 Target sequences SEQ ID Name Sequence NO ITGA6 3′UTRAUUCUUAGUCACAAAAUAUAUUUUGUUUACA 467 A SERPINE1 3′UTRAUUUUGGAGUGUAGGUGACUUGUUUACU 468 EGFR 3′UTRUAGACCCACAGACUGGUUUUGCAACGUUUAC 469 AC HIV-1 NEF siteUCCACUGACCUUUGGAUGGUGCUA 470 TET1 3′ UTR site 01 UCACUCAGUUUGGUGCUU 471TET1 3′ UTR site 02 ACUCUACACAGCUUCUGGUGCUU 472 TET1 3′ UTR site 03GUAUGGAAAACCUAAUGGUGCUU 473 SARS-CoV Site 01CUCAUGGAAAGUGAACUUGUCAUUGGUGCUG 474 SARS-CoV Site 02UCCUUCCCACAAGCAGCCCCGCAUGGUGUUG 475 SARS-CoV Site 03ACUGCCACUGCUGGAUGGACAUUUGGUGCUG 476 SARS-CoV Site 04AUUGUGGCUUAUACUAUGUCUUUAGGUGCUG 477 SARS-CoV Site 05AGACAAAUAGCGCCAGGACAAACUGGUGUUA 478 SARS-CoV Site 06UACAACAUGAGAGUUAUUCACUUUGGUGCUG 479 SARS-CoV Site 07GACCUUUUUAGAAACGCCCGUAAUGGUGUUU 480 SARS-CoV Site 08UGUAUUACACGAUGCAAUUUAGGUGGUGCUG 481 SARS-CoV Site 09ACACAUCACGAUAAAUUCACUGAUGGUGUUU 482 SARS-CoV Site 10CCACAUACAGUCUUGCAGGCUGUAGGUGCUU 483 SARS-CoV Site 11CUAGUAAGAAAAAUAUUUGUAGAUGGUGUU 484 C SARS-CoV Site 12AACAUGGACCAAGAGUCCUUUGGUGGUGCUU 485 SARS-CoV Site 13CUUAAACAACCUAAAUAGAGGUAUGGUGCUG 486 SARS-CoV Site 14UUGUCUGUUUUGCUAUCCAUGCAGGGUGCUG 487 SARS-CoV Site 15AACCGUUACUUCAGGCUUACUCUUGGUGUUU 488 SARS-CoV Site 16UUUGAUGUUGUUAGACAAUGCUCUGGUGUUA 489 SARS-CoV Site 17GCAUGCUACAAUGGUUCACCAUCUGGUGUUU 490 SARS-CoV Site 18GCUCUAAAUGACUUUAGCAACUCAGGUGCUG 491 SARS-CoV Site 19UUUACUCCUCUUGUGCAACCUGUGGGUGCUU 492 SARS-CoV Site 20UUUGGUGUACUCUUAUCUAAUUUUGGUGCUC 493 SARS-CoV Site 21AGACAACGUUCAAACCAAACACUUGGUGUUU 494 SARS-CoV Site 22CACUAUUCAGCGAGUUUCAAGAAAGGUGCUA 495 SARS-CoV Site 23CAGUUUGGUCCAACAUACUUGGAUGGUGCUG 496 SARS-CoV Site 24CCAUUGUUGUCAGCAGGCAUAUUUGGUGCUA 497 SARS-CoV Site 25UGUGGUUUUCCCUCACAGGCUGCUGGUGUUA 498 SARS-CoV Site 26GUUACCAAGGGAAAGCCCGUAAAAGGUGCUU 499 SARS-CoV Site 27AUUUUAGAAGAUGAGUUUACA 826 SARS-CoV Site 28 AUUUAGGUGGUGCUGUUUGCA 827SARS-CoV Site 29 CUUCCUUCAGGCUGUUUGC 828 SARS CoV-2 Site 01GGGCCAGAAGCUGGACUUCCCUAUGGUGCUA 500 SARS CoV-2 Site 02UCAACUCAAUUGAGUACAGACACUGGUGUUG 501 SARS CoV-2 Site 03ACUUCUGGUUGGACCUUUGGUGCAGGUGCUG 502 SARS CoV-2 Site 04UCAUAUGGUUUCCAACCCACUAAUGGUGUUG 503 SARS CoV-2 Site 05CAGGCCGGUAGCACACCUUGUAAUGGUGUUG 504 SARS CoV-2 Site 06GAUUCUUCUUCAGGUUGGACAGCUGGUGCUG 505 SARS CoV-2 Site 07AACCCUGUCCUACCAUUUAAUGAUGGUGUUU 506 SARS CoV-2 Site 08GCAUACACUAAUUCUUUCACACGUGGUGUUU 507 SARS CoV-2 Site 09UAUAAUAUGAGAGUUAUACAUUUUGGUGCU 508 G SARS CoV-2 Site 10UUCAAUUAUUAUAAGAAAGUUGAUGGUGUU 509 G SARS CoV-2 Site 11GACUUAUUUAGAAAUGCCCGUAAUGGUGUUC 510 SARS CoV-2 Site 12GCUCCAGCACAUAUAUCUACUAUUGGUGUUU 511 SARS CoV-2 Site 13AACACUGUUUACACAAAAGUUGAUGGUGUUG 512 SARS CoV-2 Site 14UGUAUAACACGUUGCAAUUUAGGUGGUGCUG 513 SARS CoV-2 Site 15AAUUCACAGACUUCAUUAAGAUGUGGUGCUU 514 SARS CoV-2 Site 16CUAGUGAGAAAAAUAUUUGUUGAUGGUGUU 515 C SARS CoV-2 Site 17CUUUCUGUUUUGCUUUCCAUGCAGGGUGCUG 516 SARS CoV-2 Site 18AACCGCUACUUUAGACUGACUCUUGGUGUUU 517 SARS CoV-2 Site 19ACAGCAAGAACUGUGUAUGAUGAUGGUGCUA 518 SARS CoV-2 Site 20GCUUGUUACAAUGGUUCACCAUCUGGUGUUU 519 SARS CoV-2 Site 21CUAAAGAGACGUGUAGUCUUUAAUGGUGUUU 520 SARS CoV-2 Site 22ACACCAGUUUACUCAUUCUUACCUGGUGUUU 521 SARS CoV-2 Site 23UUUACACCACUAAUUCAACCUAUUGGUGCUU 522 SARS CoV-2 Site 24GGCACUUGUGAAAGAUCAGAAGCUGGUGUUU 523 SARS CoV-2 Site 25GAAAACAUGACACCCCGUGACCUUGGUGCUU 524 SARS CoV-2 Site 26GUCGAAUGUACAACUAUUGUUAAUGGUGUUA 525 SARS CoV-2 Site 27AAACCAGUUACUUAUAAAUUGGAUGGUGUUG 526 SARS CoV-2 Site 28AAAGAAACUUUGUAUUGCAUAGACGGUGCUU 527 SARS CoV-2 Site 29AUACAAGAGGGUGUGGUUGAUUAUGGUGCU 528 A SARS CoV-2 Site 30CCAUUAUUAUCAGCUGGUAUUUUUGGUGCUG 529 SARS CoV-2 Site 31GUUGUAAUGGCCUACAUUACAGGUGGUGUUG 530 SARS CoV-2 Site 32AACUGGAACACUAAACAUAGCAGUGGUGUUA 531 SARS CoV-2 Site 33 ACCAUCUGGUGUUUAC829 SARS CoV-2 Site 34 GUCAGUUAGGUGGUUUACA 830 SARS CoV-2 Site 35UUUGUGCUUGCUGCUGUUUACA 831 MERS-CoV Site 01AUUAAUAGUGUGGUCCAAAAGGAUGGUGUU 532 G MERS-CoV Site 02CUUAAGCAUGGCGGUGGUAUCGCUGGUGCUA 533 MERS-CoV Site 03CAGCUUGGAUGCGUUUUCUUUAAUGGUGCUG 534 MERS-CoV Site 04AUGGCUUAUGGCAAUUGCACAUUUGGUGCUC 535 MERS-CoV Site 05CAAACCGUUCUGCAAUGUGUAAUUGGUGCUU 536 MERS-CoV Site 06CAAAUUGUCUUGCGUAAUUCUAAUGGUGCUU 537 MERS-CoV Site 07UUCACUGCUAACAAAAUUGUUGGUGGUGCUC 538 MERS-CoV Site 08GUUGCAGUAAUUGCUGGAGUUGCUGGUGCUC 539 MERS-CoV Site 09UUUAACAAGUAUAAGUACUUCUCUGGUGCUA 540 MERS-CoV Site 10CAUAUAGAACAUCCUGAUGUCUCUGGUGUUU 541 MERS-CoV Site 11UUUGUUGAAAAUCCCAGUGUUAUUGGUGUUU 542 MERS-CoV Site 12UCGCUUGGCAAAUGAGUGUGCUCAGGUGCUA 543 MERS-CoV Site 13ACUGCUAAUGUCAGUGCACUUAUGGGUGCUA 544 MERS-CoV Site 14AGCUGGAUAGGCUUCGAUGUUGAGGGUGCUC 545 MERS-CoV Site 15GUGAACUUUGUUGUUCAGCCAGUUGGUGUUG 546 MERS-CoV Site 16UCUCUGCAUCAGGUUUCCGCCUUUGGUGUUA 547 MERS-CoV Site 17GUUGUUAAACAAGGCCAUUUUAUUGGUGUUG 548 MERS-CoV Site 18GGUCCUGAUUAUGCUUACUUCAAUGGUGCUA 549 MERS-CoV Site 19ACUAUUAAAGAAAAUAUAGAUGGUGGUGCU 550 A MERS-CoV Site 20AAUUGCGUGGAAUAUUCCCUCUAUGGUGUUU 551 MERS-CoV Site 21UCCCUCUAUGGUGUUUCGGGCCGUGGUGUUU 552 MERS-CoV Site 22GCUAGCGAGCUAUCUAAUACUUUUGGUGCUA 553 MERS-CoV Site 23CUCAAAAUGGCUGGCAUGCAUUUCGGUGCUU 554 CoV-HKU1 Site 01AGUGAAGUUAAAGCCCAAUCAUCUGGUGUUA 555 CoV-HKU1 Site 02AAUCUUGAACAAAAUCAUAUUCUUGGUGUUA 556 CoV-HKU1 Site 03UUGAUAUUAAUGCGUAUGCUAAUUGGUGUU 557 G CoV-HKU1 Site 04AUAGAUGUUUUGCUUACUGUAGAUGGUGUU 558 A CoV-HKU1 Site 05GGUAUUAAACAAGAAAGUCGUGUUGGUGUU 559 G CoV-HKU1 Site 06GAUUUAGGUGUUCUUAUACAGAAUGGUGCUA 560 CoV-HKU1 Site 07GCUAGUUUUAAAGUUAUUGAUAAUGGUGUU 561 G CoV-HKU1 Site 08CCACAUCCUUAUUGUUAUUCAGAUGGUGUUA 562 CoV-HKU1 Site 09CAUUUGCAAUGGAUUGUUAUGUAUGGUGCUA 563 CoV-HKU1 Site 10UUAUUUUCAUAUUGUAGGAAAAUUGGUGUU 564 A CoV-HKU1 Site 11CCUUACACUCCAAAAUAUACUUUUGGUGUUG 565 CoV-HKU1 Site 12UCUGACGUUUAUCAACAAUUGGCUGGUGUUA 566 CoV-HKU1 Site 13AUGUAUAUUAAUACACAUAUGAUUGGUGUU 567 A CoV-HKU1 Site 14UCUUCAACAAUUAGAUUGCAGGCUGGUGUUG 568 CoV-HKU1 Site 15UAUUAUGAGUUGACUAAAAGUUGUGGUGUU 569 G CoV-HKU1 Site 16ACUUUAGUAAAAGUAGGUUUAGUUGGUGUU 570 U CoV-HKU1 Site 17CUUGUUAGACAAAUUUUUGUAGAUGGUGUUC 571 CoV-HKU1 Site 18UUGAAGAGUAUAGCAGCUACUCGUGGUGUUC 572 CoV-HKU1 Site 19AGUAUGAUGAUUUUGAGUGAUGAUGGUGUU 573 G CoV-HKU1 Site 20UUUUGAUAAAAUGAAUUAACUAAUGGUGUU 574 U CoV-HKU1 Site 21CCAUUGCAAAUAGGUUUUUCAACUGGUGUUG 575 CoV-HKU1 Site 22UGUAUCACACGAUGUAAUUUAGGUGGUGCUG 576 CoV-HKU1 Site 23GUACUUUUUGAUGGUCGUGACAAUGGUGCUU 577 CoV-HKU1 Site 24GAAGCUUUUAGAAAAGCAAGAAAUGGUGUU 578 U CoV-HKU1 Site 25AGAAAUAGUACAACAUGGAAUGGUGGUGCUU 579 CoV-HKU1 Site 26UUUACAGGUUAUUUUCCUAAAUCUGGUGCUA 580 CoV-HKU1 Site 27UAUUUUCAUUUUUAUCAAGAACGUGGUGUUU 581 CoV-HKU1 Site 28ACUCAAUCUUUUGCACCUAAUACUGGUGUUU 582 CoV-HKU1 Site 29UAUUCUAACACUGAAGUUUCUACUGGUGUUU 583 CoV-HKU1 Site 30UUACAACAAUUAUUUAAUAAAUUUGGUGCUA 584 CoV-HKU1 Site 31GACACCUCUCAUAAGAAUAAUUUUGGUGUUA 585 CoV-HKU1 Site 32CUUGCUGAAUUAGCCCCUACACCAGGUGCUU 586 HCV-1 Site 01GAUGUAUGGAGAAAAGUAUCCAGUGGUGUU 587 G HCV-1 Site 02GAAGGCAAAGACAGCUAUUGAAAUGGUGUUA 588 HCV-1 Site 03GCAUCUUGUCUGGAGAGACAAUUUGGUGUUU 589 ZEB1 3′ UTRUGUUUGAAUAUGUGGUAACAUAUGAAGGA 590 E2F1 3′ UTR UGUGCAUGUACCGGGGAAUGAAGGU591 HER3 3′ UTR UUCACAGGCACUCCUGGAGAUAUGAAGGA 592 SHIP2 3′ UTRGGGGCGGGUGUCCGUCCGGAAAUGAAGGA 593 CSNK1G1 3′ UTR SiteUGAAUUUUCAUUCAUCUUCUCAG 594 01 CSNK1G1 3′ UTR SiteAAGGUCUGAAAUUAUCUUCUCAA 595 02 CSNK1G1 3′ UTR SiteAUGUGUGGAGUUACUCUUCUCAU 596 03 CSNK1G1 3′ UTR SiteAAGCAUGGCUUGCAUUUCUCAAA 597 04 CSNK1G1 3′ UTR SiteCAGCCAAGAAAACGUCUUCUCAG 598 05 CSNK1G1 3′ UTR SiteGGUAGUUGACAUAUUUUCUCAAA 599 06 ARHGAP26 3′ UTR CGAGUUUUUGUCUUUCUUCUCAG600 Site 01 ARHGAP26 3′ UTR GAGAUACAAUCCAGUCUUCUCAU 601 Site 02ARHGAP26 3′ UTR UCUUAGAAUGUUCAGUUCUCAAU 602 Site 03 ARHGAP26 3′ UTRUCACAUACUAUUACGCUUCUCAA 603 Site 04 ARHGAP26 3′ UTRAAGUCAGCAGGAUGUCUUCUCAC 604 Site 05 RAB11FIP1 3′ UTRUGGGACCACUGUAAACUUCUCAG 605 Site 01 RAB11FIP1 3′ UTRUGUAAACCUACCCAGCUUCUCAG 606 Site 02 RAB11FIP1 3′ UTRAGACACAGGCAUGUGCUUCUCAG 607 Site 03 RBJ 3′ UTR Site 01GAAGAGAGGUUCGUACUUCUCAU 608 RBJ 3′ UTR Site 02 UUCUCAUAUAUUGAAUUCUCAAC609 SERBP1 3′ UTR Site 01 AAAAAUCUUUUUUCACUUCUCAU 610SERBP1 3′ UTR Site 02 CUACUCAAAACACAACUUCUCAG 611 CTBP1 3′ UTRGAGGCAGUUGGCAAACUUCUCAG 612 CRKL 3′ UTR AUGGAAAGGGUCUUCCUUCUCAU 613ITGA3 3′ UTR UUCUUUGUAUAUAGGCUUCUCAC 614 ITGAV 3′ UTRUGUUUUUUGUCAUUGUUCUCAAG 615 LAMC1 3′ UTR GAGUUUCCUAGUGGGCUUCUCAA 616G6PC2 3′ UTR AUGUUUUAUGUAACUCUUCUCAG 617 PPP2R5E 3′ UTRUGGACAGUAGAUGGACUUCUCAG 618 KLF17 3′ UTR GUCAGGGAAGAAAGACUUCUCAA 619MTDH 3′ UTR site 01 UUAACAACAGUGCCCUGUUUACA 621 MTDH 3′ UTR site 02AGGAACAUGGCAGUAUGUUUAC 622 MTDH 3′ UTR site 03 AACUGUCAUGGUUUAGUUUACAA623 IGF1R 3′ UTR site UUUUUUUUUUUUUUUUAGGACACC 832 UGUUUACUMET 3′ UTR site AAAGUGUUAUAUUUUUUAUAAAAA 833 UGUUUAUU IRS1 3′ UTR siteUGGUACGAUGCAUCCAUUUCAGUU 834 UGUUUACG ACVR1 ACUCAAGACUCCCACUGUG 950ASIC2 CGGGCGGGCGCCCGCGGAGG 951 ATG14 CAGCGAUGAAGAAACCG 952 ATP1A1GAGGCCUGCUUUUGAGAGGAA 953 B3GTNL1 UGGAGACUGUUUGGA 954 BANF1ACUUCCGGCGCUGUCUCGG 955 BPTF AUGAUUGUUCAGAAUA 956 CASP8AP2UGGAUGAUUGUUCAAAGA 957 CDX4 AUCUGGUUUCAGAAUC 958 CELF2AGGAUUCAGAUCUGUUUU 959 CHMP7 GCCACCUGCCGCGCAGGCGCA 960 CKMT1BACUCAAGAUUCCCAGGAG 961 CLASP1 site 01 AUAGGAUUAGAUCGGUUU 962CLASP1 site 02 CAGCCAGGAAGAAUACCU 963 CNOT3 UGGAAGAGGCCGCCUGGC 964COL15A1 CACGGAGGUCAGUUCAA 965 CYP3A4 AACAGCCUGUGCUGGCUA 966 DBET Site 01AACACCUGGCUGGCUA 967 DBET Site 02 ACGCGGGGUUGGGACGGGGUCGGGU 968DBET Site 03 CAGGCGCAACCUCUCCUAGAAA 969 DBET Site 04 CAUGAAGGGGUGGAGCCUG970 DBET Site 05 CCGGAAACAUGCAGGGAAG 971 DBET Site 06 CCGGCCUGGGAUUCCUGC972 DBET Site 07 CCGGGGAUCCCAGAGCCG 973 DBET Site 08CUCCUGGCUGCACCUGCCGCAGU 974 DBET Site 09 CUGCCUCUCCACCAGCCCA 975DBET Site 10 CUGGGAUUCCUGCCUUCU 976 DBET Site 11 GAAAUGAACGAGAGCCACA 977DBET Site 12 GCACCUGCCGCAGUGCACA 978 DBET Site 13 GCCAUUCAUGAAGGGGUG 979DBET Site 14 GCCCCGGGGGAGCUGGAG 980 DBET Site 15 GCCUGCUGCUGGAUGA 981DBET Site 16 GGCACACUCAAGACUCCCACGGAG 982 DBET Site 17GGCACCCGGAAACAUGCAGGGAA 983 DBET Site 18 GGCCGGUGAGAGACUCC 984DBET Site 19 GGCGGGAGGGAAGGCGGGCAGAGAU 985 DBET Site 20GGGCGUGUCUCCGCCCC 986 DBET Site 21 UCCAGGCCGGUGAGAGACUC 987 DBET Site 22UCCCACGGAGGUUCAGUUCCA 988 DBET Site 23 UCGGAAGAGGCGCCUCGC 989DBET Site 24 UGCGUUCCGCCGCCAGGCGCU 990 DBET site 25 CAGCGAGGAAGAAUACCG991 DCAF15 GCCCCGGGGGAGCCGGAG 992 DCN GUCUUUGAGAGGAUCCCUUU 993 DLX5AUCUGGUUUCAGAAC 994 DUSP7 GGGCCCUUCUUGAAGGGGUG 995 DUX1 GAUGGCCCUCCUGACA996 DUX4 Site 01 AACACCUGGCUGGCUA 997 DUX4 Site 02ACCUGCGCGCAGUGCGCACCCCG 998 DUX4 Site 03 ACCUUCCGACGCUGUCUAGG 999DUX4 Site 04 ACGCGGGGUUGGGACGGGGUCGGGU 1000 DUX4 Site 05AGCAGACCUGCGCGCAGUGCGCA 1001 DUX4 Site 06 AGCUCGCUGGCCUCUCUG 1002DUX4 Site 07 AGUUCAGAGAUAUAUUAAAAUGCCC 1003 DUX4 Site 08AUCUGGUUUCAGAAUC 1004 DUX4 Site 09 AUGAUUAGUUCAGAGAUA 1005 DUX4 Site 10AUGGCCCUCCCGACACCC 1006 DUX4 Site 11 CAGACCUGCGCGCAGU 1007 DUX4 Site 12CAGGCGCAACCUCUCCUAGAAA 1008 DUX4 Site 13 CCACCUUCCGACGCUG 1009DUX4 Site 14 CCGGCCUGGGAUUCCUGC 1010 DUX4 Site 15 CCGGGGAUCCCAGAGCCG1011 DUX4 Site 16 CCUCCCUGUGGAUCCUAU 1012 DUX4 Site 17CCUGCCUCUCCACCAGCCC 1013 DUX4 Site 18 CCUGCGCGCAGUGCGCACCCCG 1014GCUGACGUGC DUX4 Site 19 CCUGGAUGAUUAGUUC 1015 DUX4 Site 20CGACACCCUCGGACAGCA 1016 DUX4 Site 21 CGGAGACUCGUUUGGA 1017 DUX4 Site 22CUGGAUUAGAGUUACAUCUCCUG 1018 DUX4 Site 23 CUGGGAUUCCUGCCUUCU 1019DUX4 Site 24 CUGUGGAUCCUAUAG 1020 DUX4 Site 25 CUUCCGACGCUGUCUA 1021DUX4 Site 26 GAGCCUGCUUUGAGCGGAA 1022 DUX4 Site 27GAGCCUUUGAGAAGGAUCGCUUU 1023 DUX4 Site 28 GCCCCGGGGGAGCUGGAG 1024DUX4 Site 29 GCCUGCUGCUGGAUGA 1025 DUX4 Site 30 GGCCCGGUGAGAGACUCC 1026DUX4 Site 31 GGGAGCUCGCUGGCCUCU 1027 DUX4 Site 32 GGGCGUGUCUCCGCCCC 1028DUX4 Site 33 GUCCAGGAUUCAGAUCUGGUUU 1029 DUX4 Site 34GUGGCAGGGCGCCCGCGCAGG 1030 DUX4 Site 35 UACCAGCAGACCUGCGCGCAGU 1031DUX4 Site 36 UCCCUGUGGAUCCUAU 1032 DUX4 Site 37 UCGGAAGAGGCCGCCUCG 1033DUX4 Site 38 UCUAGGCCCGGUGAGAGACU 1034 DUX4 Site 39 UGGAUGAUUAGUUCAGAG1035 DUX4 site 40 CAGCGAGGAAGAAUACCG 1036 DUX5 Site 01 AUCUGGUUUCAGAAUC1037 DUX5 Site 02 GAUGGCCCUCCUGACA 1038 EMILIN1 AUGGCCCCCCGCACCCUC 1039EPG5 CUGUGGUUUCAGAAUC 1040 FAM13A ACUUUGAAAAGGAUCUCUUU 1041 FBX03UGGAGAUUAGAUCAGAGUG 1042 FBXL22 CCUGCGCGCACUGCGCCU 1043 FMNL3CAGAGGGGUGGAGCCUGU 1044 FREM2 CAUAAAGGGGUGGAACCUGU 1045 FRMPD2CGGAGACUUUUGGA 1046 GADD45A GUGGCAGGAGCAGCCCGC 1047 GID4CCACCCCGCGCGUGCGCGCCCCG 1048 GJD3 UGAGGCGGCGGCGCAGUGCGCCCCCG 1049 GMPRCGCCGCCCCGCGCAGGCGCCCCCGC 1050 GNAT1 CUUCGACGCUGUCA 1051 GOSR1GCCAUUCAUGAUGGUGUG 1052 GPRC6A AUGUUAGUUCAGAGAA 1053 HERC1CUGGCCUCUCCACCAGCCCUU 1054 HGF AGGAUCAGAUCUGGUUU 1055 HOOK3ACCACCAUGGCUGGCUA 1056 HOXC9 AUCUGGUUUCAGAAUC 1057 HSP40 Site 01GCCUACCGCCGCCAGGCGCU 1058 HSP40 Site 02 GGUUUGAGUUACAUCUA 1059HSP40 Site 03 UGGAGUCAGAUCUGGUUU 1060 IRF9 Site 01 CCUGGAAACAUGCAGGCAA1061 IRF9 Site 02 CGACAGCCUGGACAGCAAC 1062 IRF9 Site 03CUGGAAACAUGCAGGCAAG 1063 IRX5 UCGGAGGGCCGCCUCGAC 1064 ITGA10UCUCCCUGUGGAUCCUCAU 1065 ITGA3 Site 01 CACUGAGGUCCAGUUCCA 1066 ITGA9CUGUGGAAUCCUAUAA 1067 KCNC3 GCCUGCUGCUGGAUGA 1068 KLHL3GCCUUUGCUGCUGGAUGA 1069 KLK6 ACUCAAGAAUCCCCGGAG 1070 LARP6ACCUUCCUACGUGCUAGG 1071 MALT1 CAGUUGCCUAGACCUGGAGCAGU 1072 MAP3K4ACUCAAGCCCCACGGAG 1073 MAPK10 CACAGAGAUAUUUUAAAAUUCUU 1074 MIR4661CUGUGGAUCCUGACAGA 1075 MIR8078 Site 01 GGCCCGGUGAGAGACUCC 1076MIR8078 Site 02 UCUAGGCCCGGUGAGAGACU 1077 MTSS1 UUUGAGAAGGAUGUUUU 1078NDUFAF6 CUCACCUCUCCUAGAAA 1079 NEBL AAGAUGCAGAUCUGGUUU 1080 NKX2-3GGUAGGGACGGGGCGGGU 1081 NR2F1 CAGCGAGGAAGAAUGCCU 1082 PCID2GCCAUUCAUGAGGGGUC 1083 PDE10A CUGACCUGCCGCAGU 1084 PKD1L2AACAGAGAUAUAUGUAAAAUUGCCA 1085 PKHD1 GUCCAGGACUCACAGCUGGUUU 1086PPP1R12B GGAGGGAAGCGGGCAGAGU 1087 PTPRN2 CAGAAGGGGGGAGCCUG 1088 PYYCAGCAGACCUGCGCGCAGU 1089 RABGAPIL GGAGAUAGAGUUACAUCUU 1090 rbckCCUGCGCCCAGUGCGGACCU 1091 RFX3 UACCUUCGACACUGUCAGG 1092 RHBDF2UGGCUUCCGCCGCCAGGCCU 1093 SCRIB CUUCCAGACCUGUCUA 1094 SEMA3BUGGGACGGGGUCGCGU 1095 SETD4 GAAAUGUACCAGAGCCACA 1096 SHFLGGCUGAGCCGCGCAGUGCGGACCCUCGC 1097 SHH GGGGGCGCCCGCGCAGG 1098 SLC37A4AUGAUUAUUUUAGAGAUA 1099 SLC9A8 GGCUGGGAGGGGAAGGCGGGUCAGAGAU 1100 SMAD1GGAUUGAGAUACAUCUG 1101 SPEF1 AGAUCGCGCAGUGCGCCCCAG 1102 SPRED3CCUGCGCGCGUGCCAC 1103 ST3GAL6 GUGGCAGGCGCCGC 1104 STAG1GGUUUAGAGUUACAUUCU 1105 SUPV3L1 Site 01 CUGUGGAUCCUAUAA 1106SUPV3L1 Site 02 GGCCUGUGGAUCCUAU 1107 TBC1D26 AUGGCCUUCCUGACACCC 1108TCEA2 UCUGGGCCGGGUGAGAGAC 1109 TCF3 CUGGCUGCCCUGCGUGCAGU 1110 TM6SF1GGCUGGUUUCAGAAUC 1111 TMEM108 CUGAGACUCGUUGGA 1112 TMEM259GCCUGCUGCUGGAUGA 1113 TNFSF4 CAGGAAGGGUGGAGCCUGC 1114 TNIP1GCGGCAAGGCGCCGCGCAGG 1115 TRNP1 GAGGCCUGUUUGAGGGAA 1116 USH1GCAUCCAGGGGUGGAGCCUG 1117 WRNIP1 ACCUUCAGACGUGUCUGAGG 1118 XIAPAGAUUAGAGUUAAUCUCCC 1119 ZNF574 UUGCCUCUCCAGCAGCCCU 1120

A condition or a disease, as disclosed herein, can include a cancer, aneurological disorder, a fibrosis disease, a scarring disease, or anautoimmune disease.

In some cases, a disease or condition may comprise a neurologicaldisorder. In some cases, a neurological disorder may comprise AcquiredEpileptiform Aphasia, Acute Disseminated Encephalomyelitis,Adrenoleukodystrophy, Agenesis of the corpus callosum, Agnosia, Aicardisyndrome, Alexander disease, Alpers' disease, Alternating hemiplegia,Alzheimer's disease, Amyotrophic lateral sclerosis (see Motor NeuronDisease), Anencephaly, Angelman syndrome, Angiomatosis, Anoxia, Aphasia,Apraxia, Arachnoid cysts, Arachnoiditis, Arnold-Chiari malformation,Arteriovenous mal-formation, Asperger's syndrome, Ataxia Telangiectasia,Attention Deficit Hyperactivity Disorder, Autism, Auditory processingdisorder, Autonomic Dysfunction, Back Pain, Batten disease, Bechet'sdisease, Bell's palsy, Benign Essential Blepharospasm, Benign FocalAmyotrophy, Benign Intracranial Hypertension, Bilateral frontoparietalpolymicrogyria, Binswanger's disease, Blepharo-spasm, Bloch-Sulzbergersyndrome, Brachial plexus injury, Brain abscess, Brain damage, Brainin-jury, Brain tumor, Brown-Sequard syndrome, Canavan disease, Carpaltunnel syndrome (CTS), Causalgia, Central pain syndrome, Central pontinemyelinolysis, Centronuclear myopathy, Cephalic disorder, Cerebralaneurysm, Cerebral arteriosclerosis, Cerebral atrophy, Cerebralgigantism, Cerebral palsy, Charcot-Marie-Tooth disease, Chiarimalformation, Chorea, Chronic inflammatory de-myelinating polyneuropathy(CIDP), Chronic pain, Chronic regional pain syndrome, Coffin Lowrysyndrome, Coma, including Persistent Vegetative State, Congenital facialdiplegia, Corticobasal degeneration, Cranial arteritis,Craniosynostosis, Creutzfeldt-Jakob disease, Cumulative traumadisorders, Cushing's syndrome, Cytomegalic inclusion body disease(CIBD), Cytomegalovirus Infection, Dandy-Walker syndrome, Dawsondisease, De Morsier's syndrome, Dejerine-Klumpke palsy, Dejerine-Sottasdisease, Delayed sleep phase syndrome, Dementia, Dermatomyositis,Neurological Dyspraxia, Diabetic neuropathy, Diffuse sclerosis,Dysautonomia, Dyscalculia, Dysgraphia, Dyslexia, Dystonia, Earlyinfantile epileptic encephalopathy, Empty sella syndrome, Encephalitis,Encephalocele, Encephalotrigeminal angiomatosis, Encopresis, Epilepsy,Erb's palsy, Erythromelalgia, Essential tremor, Fabry's disease, Fahr'ssyndrome, Fainting, Familial spastic paralysis, Febrile seizures, Fishersyndrome, Friedreich's ataxia, FART Syndrome, Gaucher's disease,Gerstmann's syndrome, Giant cell arteritis, Giant cell inclusiondisease, Globoid cell Leukodystrophy, Gray matter heterotopia,Guillain-Barre syndrome, HTLV-1 associated myelopathy,Hallervorden-Spatz disease, Head injury, Headache, Hemifacial Spasm,Hereditary Spastic Paraplegia, Heredopathia atactica polyneuritiformis,Herpes zoster oticus, Herpes zoster, Hirayama syndrome,Holoprosencephaly, Huntington's disease, Hydranencephaly, Hydrocephalus,Hypercortisolism, Hypoxia, Immune-Mediated encephalomyelitis, Inclusionbody myositis, Incontinentia pigmenti, Infantile phytanic acid storagedisease, Infantile Refsum disease, Infantile spasms, Inflammatorymyopathy, Intracranial cyst, Intracranial hypertension, Joubertsyndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsboume syndrome,Klippel Feil syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru,Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffnersyndrome, Lateral medullary (Wallenberg) syndrome, Learningdisabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhansyndrome, Leukodystrophy, Lewy body dementia, Lissencephaly, Locked-Insyn-drome, Lou Gehrig's disease, Lumbar disc disease, Lymedisease—Neurological Sequelae, Macha-do-Joseph disease (Spinocerebellarataxia type 3), Macrencephaly, Maple Syrup Urine Disease,Megalencephaly, Melkersson-Rosenthal syndrome, Menieres disease,Meningitis, Menkes disease, Metachromatic leukodystrophy, Microcephaly,Migraine, Miller Fisher syndrome, Mini-Strokes, MitochondrialMyopathies, Mobius syndrome, Monomelic amyotrophy, Motor Neuron Disease,Motor skills disorder, Moyamoya disease, Mucopolysaccharidoses,Multi-Infarct Dementia, Multi-focal motor neuropathy, Multiplesclerosis, Multiple system atrophy, Muscular dystrophy, Myalgicencephalomyelitis, Myasthenia gravis, Myelinoclastic diffuse sclerosis,Myoclonic Encephalopathy of infants, Myoclonus, Myopathy, Myotubularmyopathy, Myotonia congenita, Narcolepsy, Neuro-fibromatosis,Neuroleptic malignant syndrome, Neurological manifestations of AIDS,Neurological sequelae of lupus, Neuromyotonia, Neuronal ceroidlipofuscinosis, Neuronal migration disorders, Niemann-Pick disease, Non24-hour sleep-wake syndrome, Nonverbal learning disorder,O'Sulli-van-McLeod syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara syndrome, Olivopontocerebellar atrophy,Opsoclonus myoclonus syndrome, Optic neuritis, Orthostatic Hypotension,Overuse syndrome, Palinopsia, Paresthesia, Parkinson's disease,Paramyotonia Con-genita, Paraneoplastic diseases, Paroxysmal attacks,Parry-Romberg syndrome, Rombergs Syndrome, Pelizaeus-Merzbacher disease,Periodic Paralyses, Peripheral neuropathy, Persistent Vegetative State,Pervasive neurological disorders, Photic sneeze reflex, Phytanic AcidStorage disease, Pick's disease, Pinched Nerve, Pituitary Tumors, PMG,Polio, Polymicrogyria, Polymyositis, Porencephaly, Post-Polio syndrome,Postherpetic Neuralgia (PHN), Postinfectious Encephalomyelitis, PosturalHypotension, Prader-Willi syndrome, Primary Lateral Sclerosis, Priondiseases, Progressive Hemifacial Atrophy also known as RombergsSyndrome, Progressive multifocal leukoencephalopathy, ProgressiveSclerosing Poliodystrophy, Progressive Supranuclear Palsy, Pseudotumorcerebri, Ramsay-Hunt syndrome (Type I and Type II), Rasmussen'sencephalitis, Reflex sympathetic dystrophy syndrome, Refsum disease,Repetitive motion disorders, Repetitive stress injury, Restless legssyndrome, Retrovirus-associated myelopathy, Rett syndrome, Reye'ssyndrome, Rombergs Syndrome, Rabies, Saint Vitus dance, Sandhoffdisease, Schytsophrenia, Schilder's disease, Schizencephaly, SensoryIntegration Dysfunction, Septooptic dysplasia, Shaken baby syndrome,Shingles, Shy-Drager syndrome, Sjogren's syndrome, Sleep apnea, Sleepingsickness, Snatiation, Sotos syndrome, Spasticity, Spina bifida, Spinalcord injury, Spinal cord tumors, Spinal muscular atrophy, Spinalstenosis, Steele-Richardson-Olszewski syndrome, see ProgressiveSupranuclear Palsy, Spinocerebellar ataxia, Stiff-person syndrome,Stroke, Sturge-Weber syndrome, Subacute sclerosing panencephalitis,Subcortical arteriosclerotic encephalopathy, Superficial siderosis,Syden-ham's chorea, Syncope, Synesthesia, Syringomyelia, Tardivedyskinesia, Tay-Sachs disease, Temporal arteritis, Tethered spinal cordsyndrome, Thomsen disease, Thoracic outlet syndrome, Tic Douloureux,Todd's paralysis, Tourette syndrome, Transient ischemic attack,Transmissible spongiform encephalopathies, Transverse myelitis,Traumatic brain injury, Tremor, Trigeminal neuralgia, Tropical spasticparaparesis, Trypanosomiasis, Tuberous sclerosis, Vasculitis includingtemporal arteritis, Von Hippel-Lindau disease (VHL), ViliuiskEncephalomyelitis (VE), Wallenberg's syn-drome, Werdnig-Hoffman disease,West syndrome, Whiplash, Williams syndrome, Wilson's dis-ease, X-LinkedSpinal and Bulbar Muscular Atrophy, and Zellweger syndrome. Neurologicalconditions can comprise movement disorders, for example multiple systematrophy (MSA).

In some cases, a disease or condition may comprise an autoimmunedisease. In some cases, an autoimmune disease may comprise acutedisseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, allergicasthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosingspondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome(APS), autoimmune aplastic anemia, autoimmune dysautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmunepancreatitis, autoimmune retinopathy, autoimmune thrombocytopenicpurpura (ATP), autoimmune thyroid disease, axonal & neuronalneuropathies, Balo disease, Bechet's disease, bullous pemphigoid,cardiomyopathy, Castlemen disease, celiac sprue (non-tropical), Chagasdisease, chronic fatigue syndrome, chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO),Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosalpemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease,congenital heart block, coxsackie myocarditis, CREST disease, essentialmixed cryoglobulinemia, demyelinating neuropathies, dermatomyositis,Devic's disease (neuromyelitis optica), discoid lupus, Dressler'ssyndrome, endometriosis, eosinophillic fasciitis, erythema nodosum,experimental allergic encephalomyelitis, Evan's syndrome, fibromyalgia,fibrosing alveolitis, giant cell arteritis (temporal arteritis),glomerulonephritis, Goodpasture's syn-drome, Grave's disease,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, hemolytic anemia, Henock-Schoniein purpura, herpesgestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura(ITP), IgA nephropathy, immunoregulatory lipoproteins, inclusion bodymyositis, insulin-dependent diabetes (type I), interstitial cystitis,juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eatonsyndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosis,ligneous conjunctivitis, linear IgA disease (LAD), Lupus (SLE), Lymedis-ease, Meniere's disease, microscopic polyangitis, mixed connectivetissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiplesclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica(Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis,palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), paraneoplastic cerebellardegeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Rombergsyndrome, Parsonnage-Turner syndrome, pars plantis (peripheral uveitis),pemphigus, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia, POEMS syn-drome, polyarteritis nodosa, type I, II &III autoimmune polyglandular syndromes, polymyalgia rheumatic,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, progesterone dermatitis, primary biliary cirrhosis, primarysclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathicpulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasis,Raynaud's phenomena, reflex sympathetic dystrophy, Reiter's syndrome,relapsing polychondritis, restless legs syndrome, retroperitonealfibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidtsyn-drome, scleritis, scleroderma, Slogren's syndrome, sperm andtesticular autoimmunity, stiff person syndrome, subacute bacterialendocarditis (SBE), sympathetic ophthalmia, Takayasu's arteritis,temporal arteritis/giant cell arteries, thrombocytopenic purpura (TPP),Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease (UCTD), uveitis, vasculitis,vesiculobullous dermatosis, vitiligo or Wegener's granulomatosis or,chronic active hepatitis, primary biliary cirrhosis, cadilatedcardiomyopathy, myocarditis, autoimmune polyendocrine syndrome type I(APS-I), cystic fibrosis vasculitides, acquired hypoparathyroidism,coronary artery disease, pemphigus foliaceus, pemphigus vulgaris,Rasmussen encephalitis, autoimmune gastritis, insulin hypoglycemicsyndrome (Hirata disease), Type B insulin resistance, acanthosis,systemic lupus erythematosus (SLE), pernicious anemia,treatment-resistant Lyme arthritis, polyneuropathy, demyelinatingdiseases, atopic dermatitis, autoimmune hypothyroidism, vitiligo,thyroid associated ophthalmopathy, autoimmune coeliac disease, ACTHdeficiency, dermatomyositis, Sjogren syndrome, systemic sclerosis,progressive systemic sclerosis, morphea, primary antiphospholipidsyndrome, chronic idiopathic urticaria, connective tissue syndromes,necrotizing and crescentic glomerulonephritis (NCGN), systemicvasculitis, Raynaud syndrome, chronic liver disease, visceralleishmaniasis, autoimmune C1 deficiency, membrane proliferativeglomerulonephritis (MPGN), prolonged coagulation time, immunodeficiency,atherosclerosis, neuronopathy, paraneoplastic pemphigus, paraneoplasticstiff man syndrome, paraneoplastic encephalomyelitis, subacute autonomicneuropathy, cancer-associated retinopathy, paraneoplastic opsoclonusmyoclonus ataxia, lower motor neuron syndrome and Lambert-Eatonmyasthenic syndrome.

In some cases, a disease or a condition may comprise AIDS, anthrax,botulism, brucellosis, chancroid, chlamydial infection, cholera,coccidioidomycosis, cryptosporidiosis, cyclosporiasis, dipheheria,ehrlichiosis, arboviral encephalitis, enterohemorrhagic Escherichiacoli, giardiasis, gonorrhea, dengue fever, haemophilus influenza,Hansen's disease (Leprosy), hantavirus pulmonary syn-drome, hemolyticuremic syndrome, hepatitis A, hepatitis B, hepatitis C, humanimmunodeficiency virus, legionellosis, listeriosis, Lyme disease,malaria, measles. Meningococcal disease, mumps, pertussis (whoopingcough), plague, paralytic poliomyelitis, psittacosis, Q fever, rabies,rocky mountain spotted fever, rubella, congenital rubella syndrome,shigellosis, smallpox, streptococcal disease (invasive group A),streptococcal toxic shock syndrome, Streptococcus pneumonia, syphilis,tetanus, toxic shock syndrome, trichinosis, tuberculosis, tularemia,typhoid fever, vancomycin intermediate resistant staphylocossus aureus,varicella, yellow fever, variant Creutzfeldt-Jakob dis-ease (vCJD),Ebola hemorrhagic fever, Echinococcosis, Hendra virus infection, humanmonkey-pox, influenza A, influenza B, H5N1, lassa fever, Margurghemorrhagic fever, Nipah virus, O'nyong fever, Rift valley fever,Herpes, HIV, HCV genotype 1, HCV genotype 2, HCV genotype 3, HCVgenotype 4, HCV genotype 5, HCV genotype 6, SARS-CoV-2 (COVID-19),SARS-CoV (SARS), MERS-CoV (MERS), 229E coronavirus, NL63 coronavirus,OC43 coronavirus, CoV-HKU1(HKU1), alpha coronavirus, beta coronavirus,Venezuelan equine encephalitis and West Nile virus.

In some cases, a disease or condition may comprise a fibrosis disease, ascarring disease or both. In some cases, a fibrosis disease or ascarring disease may comprise, pulmonary fibrosis, cystic fibrosis,idiopathic pulmonary fibrosis, radiation induced fibrosis, myocardialfibrosis, bridging fibrosis, cirrhosis, gliosis, arterial stiffness,arthrofibrosis, Chron's disease, Dupuytren's contracture, keloid,mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenicsystemic fibrosis, progressive massive fibrosis, retroperitonealfibrosis, scleroderma/systemic sclerosis, and adhesive capsulitis.

In some cases, a disease or condition may comprise a cancer. In somecases, a cancer may comprise thyroid cancer, adrenal cortical cancer,anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bonecancer, bone metastasis, central nervous system (CNS) cancers,peripheral nervous system (PNS) cancers, breast cancer, Castleman'sdisease, cervical cancer, childhood Non-Hodgkin's lymphoma, lymphoma,colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing'sfamily of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,gestational trophoblastic disease, hairy cell leukemia, Hodgkin'sdisease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngealcancer, acute lymphocytic leukemia, acute myeloid leukemia, children'sleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, livercancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, malebreast cancer, malignant mesothelioma, multiple myeloma, myelodysplasticsyndrome, myeloproliferative disorders, nasal cavity and paranasalcancer, nasopharyngeal cancer, neuroblastoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumor, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissuecancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer,testicular cancer, thymus cancer, uterine cancer (e.g. uterine sarcoma),vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia. Insome cases, a cancer may be selected from a list maintained by theNational Cancer Institute (https://www.cancer.gov/types).

A condition or a disease, as disclosed herein, can includehyperproliferative disorders. Malignant hyperproliferative disorders canbe stratified into risk groups, such as a low risk group and amedium-to-high risk group. Hyperproliferative disorders can include butmay not be limited to cancers, hyperplasia, or neoplasia. In some cases,the hyperproliferative cancer can be breast cancer such as a ductalcarcinoma in duct tissue of a mammary gland, medullary carcinomas,colloid carcinomas, tubular carcinomas, and inflammatory breast cancer;ovarian cancer, including epithelial ovarian tumors such asadenocarcinoma in the ovary and an adenocarcinoma that has migrated fromthe ovary into the abdominal cavity; uterine cancer; cervical cancersuch as adenocarcinoma in the cervix epithelial including squamous cellcarcinoma and adenocarcinomas; prostate cancer, such as a prostatecancer selected from the following: an adenocarcinoma or anadenocarcinoma that has mi-grated to the bone; pancreatic cancer such asepithelioid carcinoma in the pancreatic duct tissue and anadenocarcinoma in a pancreatic duct; bladder cancer such as atransitional cell carcinoma in urinary bladder, urothelial carcinomas(transitional cell carcinomas), tumors in the urothelial cells that linethe bladder, squamous cell carcinomas, adenocarcinomas, and small cellcancers; leukemia such as acute myeloid leukemia (AML), acutelymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloidleukemia, hairy cell leukemia, myelodysplasia, myeloproliferativedisorders, acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL),multiple myeloma (MM), and myelodysplastic syndrome (MDS); bone cancer;lung cancer such as non-small cell lung cancer (NSCLC), which may bedivided into squamous cell carcinomas, adenocarcinomas, and large cellundifferentiated carcinomas, and small cell lung cancer; skin cancersuch as basal cell carcinoma, melanoma, squamous cell carcinoma andactinic keratosis, which may be a skin condition that sometimes developsinto squamous cell carcinoma; eye retinoblastoma; cutaneous orintraocular (eye) melanoma; primary liver cancer (cancer that begins inthe liver); kidney cancer; autoimmune deficiency syndrome (AIDS)-relatedlympho-ma such as diffuse large B-cell lymphoma, B-cell immunoblasticlymphoma and small non-cleaved cell lymphoma; Kaposi's Sarcoma;viral-induced cancers including hepatitis B virus (HBV), hepatitis Cvirus (HCV),and hepatocellular carcinoma; human lymphotropic virus-type1 (HTLV-1) and adult T-cell leukemia/lymphoma; and human papilloma virus(HPV) and cervical cancer; central nervous system (CNS) cancers such asprimary brain tumor, which includes gliomas (astrocytoma, anaplasticastrocytoma, or glioblastoma multiforme), oligodendrogliomas,ependymomas, meningiomas, lymphomas, schwannomas, and medulloblastomas;peripheral nervous system (PNS) cancers such as acoustic neuromas andmalignant peripheral nerve sheath tumors (MPNST) including neurofibromasand schwannomas, malignant fibrous cytomas, malignant fibroushistiocytomas, malignant meningiomas, malignant mesotheliomas, andmalignant mixed Millerian tumors; oral cavity and oropharyngeal cancersuch as hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer,and oropharyngeal cancer; stomach cancer such as lymphomas, gastricstromal tumors, and carcinoid tumors; testicular cancer such as germcell tumors (GCTs), which include seminomas and nonseminomas, andgonadal stromal tumors, which include Leydig cell tumors and Sertolicell tumors; thymus cancer such as to thymomas, thymic carcinomas,Hodgkin disease, non-Hodgkin lymphomas carcinoids or carcinoid tumors;rectal cancer; and colon cancer. In some cases, the diseases stratified,classified, characterized, or diagnosed by the methods of the presentdisclosure include but may not be limited to thyroid disorders such asfor example benign thyroid disorders including but not limited tofollicular adenomas, Hurthle cell adenomas, lymphocytic thyroiditis, andthyroid hyperplasia. In some cases, the diseases stratified, classified,characterized, or diagnosed by the methods of the present disclosureinclude but may not be limited to malignant thyroid disorders such asfor example follicular carcinomas, follicular variant of papillarythyroid carcinomas, medullary carcinomas, and papillary carcinomas.

Conditions or diseases of the present disclosure can include a geneticdisorder. A genetic disorder may be an illness caused by abnormalitiesin genes or chromosomes. Genetic disorders can be grouped into twocategories: single gene disorders and multifactorial and polygenic(complex) dis-orders. A single gene disorder can be the result of asingle mutated gene. Inheriting a single gene disorder can include butnot be limited to autosomal dominant, autosomal recessive, X-linkeddominant, X-linked recessive, Y-linked and mitochondrial inheritance. Insome cases, one mutated copy of the gene can be necessary for a personto be affected by an autosomal dominant disorder. Examples of autosomaldominant type of disorder can include but are not limited toHuntington's disease, Neurofibromatosis 1, Marfan Syndrome, Hereditarynonpolyposis colorectal cancer, or Hereditary multiple exostoses. Inautosomal recessive disorders, two copies of the gene can be mutated fora subject to be affected by an autosomal recessive disorder. Examples ofthis type of disorder can include but may not be limited to cysticfibrosis, sickle-cell disease (also partial sickle-cell disease),Tay-Sachs disease, Niemann-Pick disease, or spinal muscular atrophy.X-linked dominant disorders are caused by mutations in genes on the Xchromosome such as X-linked hypophosphatemic rickets. Some X-linkeddominant conditions such as Rett syndrome, Incontinentia Pigmenti type 2and Aicardi Syndrome can be fatal. X-linked recessive disorders are alsocaused by mutations in genes on the X chromosome. Examples of this typeof disorder can include but are not limited to Hemophilia A, Duchennemuscular dystrophy, red-green color blindness, muscular dystrophy andAndrogenetic alopecia. Y-linked disorders are caused by mutations on theY chromosome. Examples can include but are not limited to MaleInfertility and hypertrichosis pinnae. The genetic disorder ofmitochondrial inheritance, also known as maternal inheritance, can applyto genes in mitochondrial DNA such as in Leber's Hereditary OpticNeuropathy.

Genetic disorders may also be complex, multifactorial or polygenic.Polygenic genetic disorders can be associated with the effects ofmultiple genes in combination with lifestyle and environmental factors.Although complex genetic disorders can cluster in families, they do nothave a clear-cut pattern of inheritance. Multifactorial or polygenicdisorders can include heart disease, diabetes, asthma, autism,autoimmune diseases such as multiple sclerosis, cancers, ciliopathies,cleft palate, hypertension, inflammatory bowel disease, mentalretardation or obesity.

Other genetic disorders can include but may not be limited to 1p36deletion syndrome, 21-hydroxylase deficiency, 22q11.2 deletion syndrome,aceruloplasminemia, achondrogenesis, type II, achondroplasia, acuteintermittent porphyria, adenylosuccinate lyase deficiency,Adrenoleu-kodystrophy, Alexander disease, alkaptonuria, alpha-1antitrypsin deficiency, Alstrom syndrome, Alzheimer's disease (type 1,2, 3, and 4), Amelogenesis Imperfecta, amyotrophic lateral sclerosis,Amyotrophic lateral sclerosis type 2, Amyotrophic lateral sclerosis type4, amyotrophic lateral sclerosis type 4, androgen insensitivitysyndrome, Anemia, Angelman syndrome, Apert syndrome,ataxia-telangiectasia, Beare-Stevenson cutis gyrata syndrome, Benjaminsyndrome, beta thalassemia, biotimidase deficiency, Birt-Hogg-Dubesyndrome, bladder cancer, Bloom syndrome, Bone diseases, breast cancer,Camptomelic dysplasia, Canavan disease, Cancer, Celiac Disease, ChronicGranulomatous Disorder (CGD), Charcot-Marie-Tooth disease,Charcot-Marie-Tooth disease Type 1, Charcot-Marie-Tooth disease Type 4,Charcot-Marie-Tooth disease Type 2, Charcot-Marie-Tooth disease Type 4,Cockayne syndrome, Coffin-Lowry syndrome, collagenopathy types II andXI, Colorectal Cancer, Congenital absence of the vas deferens,congenital bilateral absence of vas deferens, congenital diabetes,congenital erythropoietic porphyria, Congenital heart disease,congenital hypothyroidism, Connective tissue disease, Cowden syndrome,Cri du chat syndrome, Crohn's dis-ease, fibrostenosing, Crouzonsyndrome, Crouzonodermoskeletal syndrome, cystic fibrosis, De GrouchySyndrome, Degenerative nerve diseases, Dent's disease, developmentaldisabilities, Di-George syndrome, Distal spinal muscular atrophy type V,Down syndrome, Dwarfism, Ehlers-Danlos syndrome, Ehlers-Danlos syndromearthrochalasia type, Ehlers-Danlos syndrome classical type,Ehlers-Danlos syndrome dermatosparaxis type, Ehlers-Danlos syndromekyphoscoliosis type, vascular type, erythropoietic protoporphyria,Fabry's disease, Facial injuries and disorders, factor V Leidenthrombophilia, familial adenomatous polyposis, familial dysautonomia,fanconi anemia, FG syndrome, fragile X syndrome, Friedreich ataxia,Friedreich's ataxia, G6PD deficiency, galactosemia, Gaucher's disease(type 1, 2, and 3), Genetic brain disorders, Glycine encephalopathy,Haemochromatosis type 2, Haemochromatosis type 4, Harlequin Ichthyosis,Head and brain malformations, Hearing disorders and deafness, Hearingproblems in children, hemochromatosis (neonatal, type 2 and type 3),hemophilia, hepatoerythropoietic porphyria, hereditary coproporphyria,Hereditary Multiple Exostoses, hereditary neuropathy with liability topressure palsies, hereditary non-polyposis colorectal cancer,homocystinuria, Huntington's disease, Hutchinson Gilford ProgeriaSyndrome, hyperoxaluria, primary, hyperphenylalaninemia,hypochondrogenesis, hypochondroplasia, idic15, incontinentia pigmenti,Infantile Gaucher disease, infantile-onset ascending hereditary spasticparalysis, Infertility, Jackson-Weiss syndrome, Joubert syndrome,Juvenile Primary Lateral Sclerosis, Kennedy disease, Klinefeltersyndrome, Kniest dysplasia, Krabbe disease, Learning disability,Lesch-Nyhan syndrome, Leukodystrophies, Li-Fraumeni syndrome,lipoprotein lipase deficiency, familial, Male genital disorders, Marfansyndrome, McCune-Albright syndrome, McLeod syndrome, Mediterraneanfever, familial, Menkes disease, Menkes syndrome, Metabolic disorders,methemoglobinemia beta-globin type, Methemoglobinemia congenitalmethaemoglobinaemia, methylmalonic acidemia, Micro syndrome,Microcephaly, Movement disorders, Mowat-Wilson syndrome,Mucopolysaccharidosis (MPS I), Muenke syndrome, Muscular dystrophy,Muscular dystrophy, Duchenne and Becker type, muscular dystrophy,Duchenne and Becker types, myotonic dystrophy, Myotonic dystrophy type 1and type 2, Neonatal hemochromatosis, neurofibromatosis,neurofibromatosis 1, neurofibromatosis 2, Neurofibromatosis type I,neurofibromatosis type II, Neurologic diseases, Neuromuscular disorders,Niemann-Pick disease, Nonketotic hyperglycinemia, nonsyndromic deafness,Nonsyndromic deafness autosomal recessive, Noonan syn-drome,osteogenesis imperfecta (type I and type III), otospondylomegaepiphysealdysplasia, pantothenate kinase-associated neurodegeneration, PatauSyndrome (Trisomy 13), Pendred syndrome, Peutz-Jeghers syndrome,Pfeiffer syndrome, phenylketonuria, porphyria, porphyria cutanea tarda,Prader-Willi syndrome, primary pulmonary hypertension, prion disease,Progeria, propionic acidemia, protein C deficiency, protein Sdeficiency, pseudo-Gaucher disease, pseudoxanthoma elasticum, Retinaldisorders, retinoblastoma, retinoblastoma FA-Friedreich ataxia, Rettsyndrome, Rubinstein-Taybi syndrome, Sandhoff disease, sensory andautonomic neuropathy type III, sickle cell anemia, skeletal muscleregeneration, Skin pigmentation disorders, Smith Lemli Opitz Syn-drome,Speech and communication disorders, spinal muscular atrophy,spinal-bulbar muscular atrophy, spinocerebellar ataxia,spondyloepimetaphyseal dysplasia, Strudwick type, spondyloepiphysealdysplasia congenita, Stickler syndrome, Stickler syndrome COL2A1,Tay-Sachs disease, tetrahydrobiopterin deficiency, thanatophoricdysplasia, thiamine-responsive megaloblastic anemia with diabetesmellitus and sensorineural deafness, Thyroid disease, Tourette'sSyndrome, Treacher Collins syndrome, triple X syndrome, tuberoussclerosis, Turner syndrome, Usher syndrome, variegate porphyria, vonHippel-Lindau disease, Waardenburg syndrome, Weissenbacher-ZweymWllersyndrome, Wilson disease, Wolf-Hirschhom syndrome, XerodermaPigmentosum, X-linked severe combined immunodeficiency, X-linkedsideroblastic anemia, or X-linked spinal-bulbar muscle atrophy.

As used herein, a second therapy can include chemotherapy, radiation,bone marrow transplantation, immunotherapy, hormone therapy,cryotherapy, surgical procedure (such as tumor resection) or anycombination thereof. A second therapy can include administration of apharmaceutical composition, such as a small molecule. A second therapycan include administration of a pharmaceutical composition, such as oneor more antiviral drugs, for example, interferon, oseltamivir,ribavirin, daclatasvir, sofosbuvir, velpatasvir, voxilapresvir,remdesivir, indomethacin, or any combination thereof. A second therapycan include administration of a pharmaceutical composition, such as oneor more antibiotics. A second therapy can comprise administration of amuscle relaxant, an anti-depressant, a steroid, an opioid, acannabis-based therapeutic, acetaminophen, a non-steroidalanti-inflammatory, a neuropathic agent, a cannabis, a progestin, aprogesterone, or any combination thereof. A neuropathic agent maycomprise gabapentin. A non-steroidal anti-inflammatory can comprisenaproxen, ibuprofen, a COX-2 inhibitor, or any combination thereof. Asecond therapy can comprise administration of a biologic agent, cellulartherapy, regenerative medicine therapy, a tissue engineering approach, astem cell transplantation or any combination thereof. A second therapycan comprise a medical procedure. A medical procedure can comprise anepidural injection (such as a steroid injection), acupuncture, exercise,physical therapy, an ultrasound, a surgical therapy, a chiropracticmanipulation, an osteopathic manipulation, a chemonucleolysis, or anycombination thereof. A second therapy can comprise use of a breathingassist device or a ventilator. A second therapy can compriseadministration of a regenerative therapy or an immunotherapy such as aprotein, a stem cell, a cord blood cell, an umbilical cord tissue, atissue, or any combination thereof. A second therapy can comprise abiosimilar.

The term “fragment,” as used herein, can be a portion of a sequence, asubset that can be shorter than a full-length sequence. A fragment canbe a portion of a gene. A fragment can be a portion of a peptide orprotein. A fragment can be a portion of an amino acid sequence. Afragment can be a portion of an oligonucleotide sequence. A fragment canbe less than about: 20, 30, 40, 50 amino acids in length. A fragment canbe less than about: 2, 5, 10, 20, 30, 40, 50 oligonucleotides in length.

Compositions and methods as described herein include methods ofidentifying mutations or modifications to a guide strand that mayimprove therapeutic efficacy through improved knockdown (at leastpartially or completely) of one or more targets. Sequence modificationsto a passenger strand are also described herein. Chemical modificationsto one or more both strands are also described herein. Constructs ormimics as described herein may comprise one or more sequence alterationsin a guide strand that may curb target recognition toward a preferredmRNA target among a plurality of mRNA targets. Such constructs or mimicsmay enhance stability, at least partially reduce or eliminate an immunestimulation, improve a pharmacological activity, retain one or morepoly-targeting effect, or any combination thereof.

Compositions and methods as described herein can include miR mimics(such as miR-30, miR-29, miR-26, miR-27, miR-101, miR-145, miR-205,miR-338, and miR-375 mimics) having improved stability, safety, and/oractivity as compared to a comparable miR (such as miR-30, miR-29,miR-26, miR-27, miR-101, miR-145, miR-205, miR-338, and miR-375).

Mimics can be identified using intelligent design, testing of one ormore chemical modification patterns, sequence mutation in passengerstrands that can improve mimic complementarity or alter mimic duplexstructure, and sequence mutation to the guide strand that can yieldbenefits in activity. In some cases, a sequence mutation in the guidestrand can move beyond use of natural guide sequence to use of mimics.For example, sequence alterations can tailor guide strand activitytoward preferred downstream targets, such as clinical targets.

Because miRNAs are grouped in families based on sequence similarity,mutant sequences may be ‘non-natural’ or ‘artificial’ miRNA familymembers.

The miR-30 family of microRNAs (miRNAs) can include 5 family members inhumans (i.e. miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d, andmiR-30e). This family can possess tumor suppressor activity in severaltypes of cancers and suppression of fibrosis, scarring or both. Forexample, miR-30 downregulation can be linked to poor outcome inrecurrent head and neck cancer patients. As such, the miR-30 family isan ideal candidate for miRNA replacement therapy. However, developingmiR-30 mimics into novel drug candidates can include overcomingpharmacological barriers for therapeutic application such as: (i)inherent metabolic instability being readily degraded by a variety ofnucleases found in biological fluids; (ii) contain sequence patterns(e.g. CpG motifs) and structures that may be natural ligands for cellreceptors (i.e. TLR family, RIG-I-like receptors, and other RNA sensors)that trigger the innate immune response that can lead to toxicities invivo; (iii) poor pharmacology in regards to potency and/or efficacy asnatural miRNA sequences may not inherently optimized for mimetics; (iv)delivery in vivo to diseased or target tissues; (v) or any combinationthereof.

The miR-29 family of microRNAs (miRNAs) can include 4 family members inhumans (i.e. miR-29a, miR-29b-1, miR-29b-2, and miR-29c). This familycan possess tumor suppressor activity in several types of cancers,antiviral activity, and suppression of fibrosis, scarring or both. Forexample, miR-29 overexpression can be linked to reduce Hepatitis C Viralabundance in cell culture. As such, the miR-29 family is an idealcandidate for miRNA replacement therapy. However, developing miR-29mimics into novel drug candidates can include overcoming pharmacologicalbarriers for therapeutic application such as: (i) inherent metabolicinstability being readily degraded by a variety of nucleases found inbiological fluids; (ii) contain sequence patterns (e.g. CpG motifs) andstructures that may be natural ligands for cell receptors (i.e. TLRfamily, RIG-I-like receptors, and other RNA sensors) that trigger theinnate immune response that can lead to toxicities in vivo; (iii) poorpharmacology in regards to potency and/or efficacy as natural miRNAsequences may not inherently optimized for mimetics; (iv) delivery invivo to diseased or target tissues; (v) or any combination thereof.

In addition, high-degree of chemical modification and/or implementingconventional modification patterns based on siRNA design can oftennegatively impact miRNA mimic activity by reducing their pleiotropiceffects of silencing multiple transcripts. Chemical modifications canalter sequence recognition of otherwise natural downstream targets.While desirable for improving siRNA specificity towards its intendedtarget by reducing its “miRNA-like” off-target effect, it can beundesirable for miRNA mimetics that are developed to deliberately targetmultiple transcripts.

An oligonucleotide can comprise a sugar modification. An oligonucleotidecan comprise a plurality of sugar modifications. A sugar modificationcan comprise a glucose or derivative thereof. A sugar modification cancomprise a ribose or deoxyribose. A sugar modification can comprise amonosaccharide, a disaccharide, a trisaccharide or any combinationthereof.

An oligonucleotide can comprise a chemical modification. Anoligonucleotide can comprise a plurality of chemical modifications. Anoligonucleotide can comprise a plurality of chemical modificationswithin a portion of an oligonucleotide, such as a terminal end. Achemical modification can comprise a methyl group, a fluoro group, amethoxyethyl group, an ethyl group, an amide group, an ester group, morethan one of any of these, or any combination thereof. A chemicalmodification can comprise 1-methyl-adenosine, 1-methyl-guanosine,1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine,2′-amino-2′-deoxyadenosine, 2′-amino-2′-deoxycytidine,2′-amino-2′-deoxyguanosine, 2′-amino-2′-deoxyuridine,2-amino-6-chloropurineriboside, 2-aminopurine-riboside, 2′-araadenosine,2′-aracytidine, 2′-arauridine, 2′-azido-2′-deoxyadenosine,2′-azido-2′-deoxycytidine, 2′-azido-2′-deoxyguanosine,2′-azido-2′-deoxyuridine, 2-chloroadenosine,2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxycytidine,2′-fluoro-2′-deoxyguanosine, 2′-fluoro-2′-deoxyuridine,2′-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-methyl-2-aminoadenosine,2′-O-methyl-2′-deoxyadenosine, 2′-O-methyl-2′-deoxycytidine,2′-O-methyl-2′-deoxyguanosine, 2′-O-methyl-2′-deoxyuridine,2′-O-methyl-5-methyluridine, 2′-O-methylinosine,2′-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine,3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine,5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine,5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine,5-carboxymethylaminomethyl-2-thio-uracil,5-carboxymethylamonomethyl-uracil, 5-chloro-ara-cytosine,5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine,5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-azauridine,6-chloro-7-deaza-guanosine, 6-chloropurineriboside,6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside,7-deaza-2′-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine,8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine,8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside,beta-D-mannosyl-queosine, dihydro-uridine, inosine, N1-methyladenosine,N6-([6-ami nohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine,N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acidmethyl ester, puromycin, queosine, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine,xylo-adenosine, or any combination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicalmodification, such as a chemical modification to a base or a sugar ofthe engineered oligonucleotide, relative to a natural base or sugar. Insome cases, the engineered oligonucleotide can comprise more than onechemical modification, such as a plurality of chemical modifications. Aportion of bases or a portion of sugars of the engineeredoligonucleotide can comprise one or more chemical modifications. In somecases, about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ofbases or sugars in an engineered oligonucleotide can be chemicallymodified.

In some cases, a ribonucleotide (or in some cases a deoxynucleotide),can be modified, such as the base component, the sugar (ribose)component, the phosphate component forming the backbone of theengineered oligonucleotide, or any combination thereof, by a chemicalmodification as described herein.

In some cases, an engineered oligonucleotide can be engineered ormodified to increase a specificity for an RNA sequence among a pluralityof RNA sequences. An engineered oligonucleotide can be modified tosignificantly increase a specificity for an RNA sequence among aplurality of RNA sequences. Increased specificity can be compared to acomparable oligonucleotide that may not be engineered or can be comparedto a comparable oligonucleotide that may be engineered or modified in adifferent way. A specificity may be increased by at least about: 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more as compared to a comparable oligonucleotide.An engineered oligonucleotide can be engineered or modified to increasea specificity for a first RNA sequence as compared to a second RNAsequence

In some cases, an engineered oligonucleotide can be selective for an RNAsequence encoding a gene, such as a gene implicated in a disease orcondition as described herein, such as an oncogene. In some cases, theengineered oligonucleotide can be selective for an RNA sequence encodingan oncogene, among a plurality of RNA sequences. In some cases, anengineered oligonucleotide can comprise an increased specificity for anRNA sequence encoding a gene, such as an oncogene, among a plurality ofRNA sequences. In some cases, an engineered oligonucleotide can beselective for an RNA sequence encoding ITGA6, SERPINE1, EGFR, or anycombination thereof, among a plurality of RNA sequences.

A chemically modified nucleotide as used herein can be guanosine,uridine, adenosine, thymidine or cytosine including, without implyingany limitation, any natively occurring or non-natively occurringguanosine, uridine, adenosine, thymidine or cytidine that has beenaltered chemically, for example by acetylation, methylation,hydroxylation, etc., including 1-methyl-adenosine, 1-methyl-guanosine,1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine,2′-amino-2′-deoxyadenosine, 2′-amino-2′-deoxycytidine,2′-amino-2′-deoxyguanosine, 2′-amino-2′-deoxyuridine,2-amino-6-chloropurineriboside, 2-aminopurine-riboside, 2′-araadenosine,2′-aracytidine, 2′-arauridine, 2′-azido-2′-deoxyadenosine,2′-azido-2′-deoxycytidine, 2′-azido-2′-deoxyguanosine,2′-azido-2′-deoxyuridine, 2-chloroadenosine,2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxycytidine,2′-fluoro-2′-deoxyguanosine, 2′-fluoro-2′-deoxyuridine,2′-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-methyl-2-aminoadenosine,2′-O-methyl-2′-deoxyadenosine, 2′-O-methyl-2′-deoxycytidine,2′-O-methyl-2′-deoxyguanosine, 2′-O-methyl-2′-deoxyuridine,2′-O-methyl-5-methyluridine, 2′-O-methylinosine,2′-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine,3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine,5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine,5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine,5-carboxymethylaminomethyl-2-thio-uracil,5-carboxymethylamonomethyl-uracil, 5-chloro-ara-cytosine,5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine,5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-azauridine,6-chloro-7-deaza-guanosine, 6-chloropurineriboside,6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside,7-deaza-2′-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine,8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine,8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside,beta-D-mannosyl-queosine, dihydro-uridine, inosine, N1-methyladenosine,N6-([6-ami nohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine,N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxy acetic acidmethyl ester, puromycin, queosine, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, orxylo-adenosine. The preparation of such variants is known to the personskilled in the art, for example from U.S. Pat. Nos. 4,373,071,4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679,5,047,524, 5,132,418, 5,153,319, 5,262,530 or 5,700,642.

In some cases, the engineered nucleotide can comprise a chemicallymodified nucleotide such as2-amino-6-chloropurineriboside-5′-triphosphate,2-aminopurine-riboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate,2′-amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-fluorothymidine-5′-triphosphate,2′-O-methyl-inosine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-ami noallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-propynyl-2′-deoxycytidine-5′-triphosphate,5-propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, puromycin-5′-triphosphate,xanthosine-5′-triphosphate, or any combination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide such as pyridin-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, or anycombination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide such as 5-aza-cytidine, pseudoisocytidine,3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, orany combination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide such as 2-aminopurine, 2,6-diaminopurine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, or anycombination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide such as inosine, 1-methyl-inosine, wyosine,wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine,6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, or anycombination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide such as 6-aza-cytidine, 2-thio-cytidine,alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine,alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine,6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine, or any combination thereof.

In some cases, the engineered oligonucleotide can comprise a chemicallymodified nucleotide, which can be chemically modified at the 2′position. The chemically modified oligonucleotide can comprise asubstituent at the 2′ carbon atom, wherein the substituent can comprisea halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino groupor an aminoalkoxy group, such as a 2′-hydrogen (2′-deoxy), 2′-O-methyl,2′-O-methoxyethyl, 2′-fluoro, 2′ Methoxyethyl, 2′-fluoro, Locked Nucleicacid, or any combination thereof.

Another chemical modification (such as one involving the 2′ position ofa nucleotide) can be a locked nucleic acid (LNA) nucleotide, an ethylenebridged nucleic acid (ENA) nucleotide, an (S)-constrained ethyl cEtnucleotide, a bridged nucleic acid (BNA) or any combination thereof. Abackbone modification can lock the sugar of the modified nucleotide intoa preferred northern conformation. In some case, a presence of that typeof modification in the target sequence of the engineered oligonucleotidecan allow for stronger and faster binding of the targeting sequence tothe target site.

In some cases, the engineered oligonucleotide can comprise at least onechemically modified nucleotide, wherein the phosphate backbone, whichcan be incorporated into the engineered oligonucleotide, can bemodified. One or more phosphate groups of the backbone can be modified,for example, by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleotide can include afull replacement of an unmodified phosphate moiety with a modifiedphosphate as described herein. Examples of modified phosphate groups caninclude a phosphorothioate, a methylphosphonate, a phosphoroselenate, aborano phosphate, a borano phosphate ester, a hydrogen phosphonate, aphosphoroamidate, an alkyl phosphonate, an aryl phosphonate or aphosphotriester. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

In some cases, the engineered oligonucleotide can comprise a sugarmodification. The sugar modification can comprise a conjugate, such as alinker. In some cases, the engineered oligonucleotide can comprise oneor more linker groups. The engineered oligonucleotide can be linked toan antibody, a protein, a lipid, an aptamer, a small molecule, a drug,or any combination thereof. A linker can form a covalent bond. Theengineered oligonucleotide can be linked to one or more engineeredoligonucleotides, such as a second engineered oligonucleotide via alinker. In some cases, the linker may be a cleavable linker. In somecases, a linker can comprise an azide linker. An engineeredoligonucleotide can comprise a base of a nucleotide that is glycosylatedwith a glycan. In some cases, the engineered oligonucleotide cancomprise an abasic site, such as a nucleotide lacking an organic base.In some cases, the abasic nucleotide can comprise a chemicalmodification as described herein, such as at the 2′ position of theribose. In some cases, the 2′ C atom of the ribose can be substitutedwith a substituent such as a halogen, an alkoxy group, a hydrogen, anaryloxy group, an amino group or an aminoalkoxy group, in some casesfrom 2′-hydrogen (2′-deoxy), 2′-O-methyl, 2′-O-methoxyethyl or2′-fluoro. In some cases, an abasic site nucleotide can comprisestructures 1 A or 1 B:

In some cases, the engineered oligonucleotide can be modified by theaddition of a ‘5’-CAP’ structure. A 5′-cap can be an entity, such as amodified nucleotide entity, which can ‘cap’ the 5′-end of a maturemiRNA. A 5′-cap can typically be formed by a modified nucleotide,particularly by a derivative of a guanine nucleotide. In some cases, the5′-cap can be linked to the 5′-terminus of the engineeredoligonucleotide via a 5′-51-triphosphate linkage. A 5′-cap can bemethylated, e.g. m7GpppN, wherein N can be the terminal 5′ nucleotide ofthe nucleic acid carrying the 5′-cap, such as the 5′-end of an RNA. A5′-cap structure can include glyceryl, inverted deoxy abasic residue(moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl)nucleotide, 4′-thio nucleotide, carbocyclic nucleotide,1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modifiedbase nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxy pentyl nucleotide, 3′-3′-inverted nucleotide moiety,3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety,3′-2′-inverted abasic moiety, 1,4-butanediol phosphate,3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,3′phosphorothioate, phosphorodithioate, or bridging or non-bridgingmethylphosphonate moiety. In some cases, a modified 5′-CAP structure cancomprise a CAP1 (methylation of the ribose of the adjacent nucleotide ofm7G), CAP2 (methylation of the ribose of the 2nd nucleotide downstreamof the m7G), CAP3 (methylation of the ribose of the 3rd nucleotidedownstream of the m7G), CAP4 (methylation of the ribose of the 4thnucleotide downstream of the m7G), ARCA (anti-reverse CAP analogue,modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.

The term “epigenetic marker” as used herein, can be any covalentmodification of a nucleic acid base. In some cases, a covalentmodification can comprise adding a methyl group, a hydroxymethyl group,a carbon atom, an oxygen atom, or any combination thereof to one or morebases of a nucleic acid sequence. In some cases, a covalent modificationcan comprise changing an oxidation state of a molecule associated with anucleic acid sequence, such as an oxygen atom, or a combination thereof.A covalent modification can occur at any base, such as a cytosine, athymine, a uracil, an adenine, a guanine, or any combination thereof. Insome cases, an epigenetic modification can comprise an oxidation or areduction. A nucleic acid sequence can comprise one or moreepigenetically modified bases. An epigenetically modified base cancomprise any base, such as a cytosine, a uracil, a thymine, adenine, ora guanine. An epigenetically modified base can comprise a methylatedbase, a hydroxymethylated base, a formylated base, or a carboxylic acidcontaining base or a salt thereof. An epigenetically modified base cancomprise a 5-methylated base, such as a 5-methylated cytosine (5-mC). Anepigenetically modified base can comprise a 5-hydroxymethylated base,such as a 5-hydroxymethylated cytosine (5-hmC). An epigeneticallymodified base can comprise a 5-formylated base, such as a 5-formylatedcytosine (5-fC). An epigenetically modified base can comprise a5-carboxylated base or a salt thereof, such as a 5-carboxylated cytosine(5-caC). In some cases, an epigenetically modified base can comprise amethyltransferase-directed transfer of an activated group (mTAG).

An epigenetically modified base can comprise one or more bases or apurine (such as Structure 1) or one or more bases of a pyrimidine (suchas Structure 2). An epigenetic modification may occur at one or more ofany positions. For example, an epigenetic modification can occur at oneor more positions of a purine, including positions 1, 2, 3, 4, 5, 6, 7,8, 9, as shown in Structure 1. In some cases, an epigenetic modificationcan occur at one or more positions of a pyrimidine, including positions1, 2, 3, 4, 5, 6, as shown in Structure 2.

A nucleic acid sequence may comprise an epigenetically modified base. Anucleic acid sequence may comprise a plurality of epigeneticallymodified bases. A nucleic acid sequence may comprise an epigeneticallymodified base positioned within a CG site, a CpG island, or acombination thereof. A nucleic acid sequence may comprise differentepigenetically modified bases, such as a methylated base, ahydroxymethylated base, a formylated base, a carboxylic acid containingbase or a salt thereof, a plurality of any of these, or any combinationthereof. In some cases, an engineered oligonucleotide or salt thereof,when chemically modified, can be of formula: Guide Pattern 1, GuidePattern 2, or Guide Pattern 3 as shown in Table 7.

TABLE 7 Chemical modification patterns Pattern Sequence (5′-3′)Guide Pattern 1 (N)_(a)(mN)_(b)(N)cNN Guide Pattern 2(N)_(a)(mN)_(b)(N)_(c)sfNsmN Guide Pattern 3(fNmN)_(h)(mN)_(i)(fNmN)_(j)sfNsmN Passenger CAP-mNmNmN(N)_(k)mNmNmNPattern 1 N can be any natural or non-natural nucleotide; mN can be a2′-O-methyl-modified uracil, guanine, adenine, or cytosine; s can be aphosphothionate-modified backbone; fN can be 2′fluoro-modified uracil,guanine, adenine, or cytosine; CAP can be 5′-terminal methyl group(5′-OMethyl) or alkylamino group such as amino-carbon 6 chain (5′-AminoC6); a can be from 8-10; b can be from 7-10; c can be from 2-4; h can be5-7; i can be 0 or 1; j can be 3-4; and k can be 12-19.

An engineered oligonucleotide or salt thereof, when chemically modified,can have at least 90% sequence identity to any one of SEQ ID NOs: 52-89,127-154, 184-201, 205-222, 225-233, 235-243, 264-443, 445-453, 455-463,620, 644-825, 835-837, and 846-899.

In some cases, a vector can be used for miR delivery, in an in vitrosetting, in vivo setting, or any combination thereof. In some cases, thevector can be targeted to but may not be limited to a mammal, or aspecific organ, or a specific cell, or any combination thereof. Thevector can comprise any composition described herein. In some cases, thevector can comprise more than one composition, such as a miR-30 familyconstruct and a second miR-29 family construct, or a miR-30 familyconstruct and a second miR-30 family construct, or a miR-29 familyconstruct and a second miR-29 family construct, or any combinationthereof. In some cases, the vector can be comprised of a liposome, ananoparticle or any combination thereof. The liposome can include butmay not be limited to unilamellar liposome, multilamellar liposome,archaeosome, niosome, novasome, cryptosome, emulsome, vesosome, or aderivative of any of these, or any combination thereof. The nanoparticlecan include but may not be limited to biopolymeric nanoparticle,alginate nanoparticle, xanthan gum nanoparticle, cellulose nanoparticle,dendrimer, polymeric micelle, polyplexed, inorganic nanoparticle,nanocrystal, metallic nanoparticle, quantum dot, protein nanoparticle,polysaccharide nanoparticle, or a derivative of any of these, or anycombination thereof. In some cases, the vector can be an RNA viralvector which can include but may not be limited to a retrovirus,lentivirus, coronavirus, alphavirus, flavivirus, rhabdovirus,morbillivirus, picornavirus, coxsackievirus, or picornavirus or portionsof any of these, or fragments of any of these, or any combinationthereof. In some cases, the vector can be a DNA viral vector which caninclude but may not be limited to an adeno-associated viral (AAV)vector, adenovirus, hybrid adenoviral system, hepadnavirus, parvovirus,papillomavirus, polyomavirus, herpesvirus, poxvirus, a portion of any ofthese, or a fragment of any of these, or any combination thereof.

Compositions and methods are described herein can include miR mimics.Such miR mimics can contain one or more sequence modifications, one ormore chemical modifications, or a combination thereof that can: enhancestability of the miR mimic; substantially reduce or eliminate immunestimulation (such as via the innate immune response); improvepharmacological activity of the miR mimic; retain poly-targeting effectsof the miR mimic; or any combination thereof. Engineered miR familymembers comprising sequence alterations in the guide strand can biastarget recognition toward two or more clinical targets.

As used herein, a “biosimilar” or a “biosimilar product” can refer to abiological product that is licensed based on a showing that it issubstantially similar to an FDA-approved biological product, known as areference product, and has no clinically meaningful differences in termsof safety and effectiveness from the reference product. Only minordifferences in clinically inactive components may be allowable inbiosimilar products. A “biosimilar” of an approved referenceproduct/biological drug refers to a biologic product that is similar tothe reference product based upon data derived from (a) analyticalstudies that demonstrate that the biological product is highly similarto the reference product notwithstanding minor differences in clinicallyinactive components; (b) animal studies (including the assessment oftoxicity); and/or (c) a clinical study or studies (including theassessment of immunogenicity and pharmacokinetics or pharmacodynamics)that are sufficient to demonstrate safety, purity, and potency in one ormore appropriate conditions of use for which the reference product islicensed and intended to be used and for which licensure is sought forthe biological product. In some embodiments, the biosimilar biologicalproduct and reference product utilize the same mechanism or mechanismsof action for the condition or conditions of use prescribed,recommended, or suggested in the proposed labeling, but only to theextent the mechanism or mechanisms of action are known for the referenceproduct. In some embodiments, the condition or conditions of useprescribed, recommended, or suggested in the labeling proposed for thebiological product have been previously approved for the referenceproduct. In some embodiments, the route of administration, the dosageform, and/or the strength of the biological product are the same asthose of the reference product. In some embodiments, the facility inwhich the biological product is manufactured, processed, packed, or heldmay meet standards designed to assure that the biological productcontinues to be safe, pure, and potent. The reference product may beapproved in at least one of the U.S., Europe, or Japan. In someembodiments, a response rate of human subjects administered thebiosimilar product can be 50%-150% of the response rate of humansubjects administered the reference product. For example, the responserate of human subjects administered the biosimilar product can be50%-100%, 50%-110%, 50%-120%, 50%-130%, 50%-140%, 50%-150%, 60%-100%,60%-110%, 60%-120%, 60%-130%, 60%-140%, 60%-150%, 70%-100%, 70%-110%,70%-120%, 70%-130%, 70%-140%, 70%-150%, 80%-100%, 80%-110%, 80%-120%,80%-130%, 80%-140%, 80%-150%, 90%-100%, 90%-110%, 90%-120%, 90%-130%,90%-140%, 90%-150%, 100%-110%, 100%-120%, 100%-130%, 100%-140%,100%-150%, 110%-120%, 110%-130%, 110%-140%, 110%-150%, 120%-130%,120%-140%, 120%-150%, 130%-140%, 130%-150%, or 140%-150% of the responserate of human subjects administered the reference product. In someembodiments, a biosimilar product and a reference product can utilizethe same mechanism or mechanisms of action for the condition orconditions of use prescribed, recommended, or suggested in the proposedlabeling, but only to extent the mechanism or mechanisms are known forthe reference product. To obtain approval for biosimilar drugs, studiesand data of structure, function, animal toxicity, pharmacokinetics,pharmacodynamics, immunogenicity, and clinical safety and efficacy maybe needed. A biosimilar may also be known as a follow-on biologic or asubsequent entry biologic. In some embodiments, a biosimilar product maybe substantially similar to the reference product notwithstanding minordifferent in clinically inactive components.

As used herein, a “interchangeable biological product” may refer to abiosimilar of an FDA-approved reference product and may meet additionalstandards for interchangeability. In some embodiments, aninterchangeable biological product can, for example, produce the sameclinical result as the reference product in any given subject. In someembodiments, an interchangeable product may contain the same amount ofthe same active ingredients, may possess comparable pharmacokineticproperties, may have the same clinically significant characteristics,and may be administered in the same way as the reference compound. Insome embodiments, an interchangeable product can be a biosimilar productthat meets additional standards for interchangeability. In someembodiments, an interchangeable product can produce the same clinicalresult as a reference product in all the reference product's licensedconditions of use. In some embodiments, an interchangeable product canbe substituted for the reference product by a pharmacist without theintervention of the health care provider who prescribed the referenceproduct. In some embodiments, when administered more than once to anindividual, the risk in terms of safety or diminished efficacy ofalternating or switching between use of the biological product and thereference product is not greater than the risk of using the referenceproduct without such alternation or switch. In some embodiments, aninterchangeable product can be a regulatory agency approved product. Insome embodiments, a response rate of human subjects administered theinterchangeable product can be 80%-120% of the response rate of humansubjects administered the reference product. For example, the responserate of human subjects administered the interchangeable product can be80%-100%, 80%-110%, 80%-120%, 90%-100%, 90%-110%, 90%-120%, 100%-110%,100%-120%, or 110%-120 of the response rate of human subjectsadministered the reference product.

In some cases, at least a portion of the RNA sequence can be encoded byan oncogene. The oncogene can comprise ABL1, ABL2, AKT1, AKT2, AKT3,ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF, CARD11, CBLB, CBLC, CCND1,CCND2, CCND3, CDX2, CTNNB1, DDB2, BBIT3, BBX6, DEK, EGFR, ELK4, ERBB2,ERBB3, E2F1, ETV4, ETV6, EVIl, EWSR1, FEV, FGFR1, FGFR1OP, FGR2, FUS,GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, ITGA6, JUN, KIT, KRAS, LCK,LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MLL, MPL, MYB, MYC, MYCL1,MYCN, NCOA4, NFKB2, NRAS, NTRKI, NUP214, PAX8, PDGFB, PIK3CA, PIM1,PLAGI, PPARG, PTPN11, RAF1, REL, RET, ROS1, SERPINE1, SMO, SS18, TCL1A,TET2, TFG, TLX1, TPR, USP6, or any combination thereof. In some cases,the oncogene can comprise ITGA6, BCL2, DEK, PLAGI, SERPINE1, MYCN, LMO2,PIM1, EGFR, IRS1, NT5E, EGFR, GLDC, SOCS1, STAT1, LOX, PDGFRB, WNT5A,CD80, CCNA1, THBS2, IGF1R, AFAP1L2, CTHRC1, MET, FAP, SERPINE1, ILIA,GJA1, MYBL2, CDK6, ATG9A, SETDBlor any combination thereof. In somecases, an engineered oligonucleotide or salt thereof as described hereincan be selective for one or more particular RNA sequences among aplurality of RNA sequences. For example, an engineered oligonucleotideor salt thereof as described herein can be selective for one or more ofABL1, ABL2, AKT1, AKT2, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF,CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2, BBIT3,BBX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVIl, EWSR1, FEV, FGFR1,FGFR1OP, FGR2, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, ITGA6, JUN,KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MLL, MPL, MYB,MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB,PIK3CA, PIM1, PLAGI, PPARG, PTPN11, RAF1, REL, RET, ROS1, SERPINE1, SMO,SS18, TCL1A, TET2, TFG, TLX1, TPR, USP6, relative to a plurality ofother RNA sequences.

A portion of an RNA sequence can be at least about: 70%, 75%, 80%, 85%,90%, 95% of the bases of an RNA sequence.

In some cases, the disease or condition can comprise fibrosis. In somecases, at least a portion of the RNA sequence can be encoded by acollagen super family gene, a platelet-derived growth factor gene, aTGF-0 signaling gene, a collagen remodeling gene, an extracellularmatrix remodeling gene, a Wnt signaling gene, a hepatoma-derived growthfactor (HDGF) signaling gene, or any combination thereof. In some cases,at least a portion of the RNA sequence can be encoded by COL1A1,COL11A1, COL2A1, COL5A3, COL5A2, COL4A4, COL21A1, COL7A1, COL9A1,COL19A1, COL5A1, COL22A1, COL8A1, COL4A2, COL6A2, COL24A1, COL4A3,COL4A6, COL25A1, COL16A1, COL15A1, or any combination thereof. In somecases, at least a portion of the RNA sequence can be encoded by theplatelet-derived growth factor gene, such as PDGFB, PDGFC, or PDGFRB. Insome cases, at least a portion of the RNA sequence can be encoded by theTGF-β signaling gene (such as WISP1 or TGFB2). In some cases, at least aportion of the RNA sequence can be encoded by the collagen remodelinggene (such as LOXL2). In some cases, at least a portion of the RNAsequence can be encoded by the extracellular matrix remodeling gene(such as COL1A1, COL11A1, COL2A1, COL5A3, COL5A2, COL4A4, COL21A1,COL7A1, COL9A1, COL19A1, COL5A1, COL22A1, COL8A1, COL4A2, COL6A2,COL24A1, COL4A3, COL4A6, COL25A1, COL16A1, COL15A1, LOXL2, or Elastin).In some cases, at least a portion of the RNA sequence can be encoded bythe Wnt signaling gene (such as WISP1). In some cases, at least aportion of the RNA sequence can be encoded by the HDGF signaling gene(such as HDGF). A portion can be at least about: 60%, 70%, 80%, 90%, 95%of the bases on the RNA sequence.

In some cases, the engineered oligonucleotide or salt thereof can befrom about 5 to about 50 nucleotides in length. In some cases, theengineered oligonucleotide or salt thereof can be from about 5 to about40 nucleotides in length. In some cases, the engineered oligonucleotideor salt thereof can be from about 5 to about 30 nucleotides in length.In some cases, the engineered oligonucleotide or salt thereof can befrom about 5 to about 25 nucleotides in length. In some cases, theengineered oligonucleotide or salt thereof can be from about 5 to about60 nucleotides in length. In some cases, the engineered oligonucleotideor salt thereof can be from about 5 to about 80 nucleotides in length.In some cases, the engineered oligonucleotide or salt thereof can befrom about 5 to about 100 nucleotides in length. In some cases, theengineered oligonucleotide or salt thereof can be from about 5 to about200 nucleotides in length.

In some cases, the engineered oligonucleotide or salt thereof, whencontacted with the mRNA sequence, can produce at least about: 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 fold lowerexpression of a polypeptide encoded by the mRNA sequence, as compared tocontacting an equivalent amount of the otherwise comparableoligonucleotide with the mRNA sequence. Lower expression can be fromabout 1.2-fold to about 10-fold lower expression.

In some cases, the engineered oligonucleotide or salt thereof, whencontacted with the mRNA sequence, can produce at least about: 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9,10 fold loweractivity of a polypeptide encoded by the mRNA sequence, as compared tocontacting an equivalent amount of the otherwise comparableoligonucleotide with the mRNA sequence. Lower activity can be from about1.2-fold to about 10-fold lower activity.

In some cases, at least about: 70%, 7%, 80%, 85%, 90%, 95% of an initialamount of the engineered oligonucleotide or salt thereof remains whenthe engineered oligonucleotide or salt thereof can be stored in a closedcontainer placed in a room for a time period of at least about: 1 month,2 months, 3 months, 4 months, 5 months, 6 months at about from about 21to about 25 degrees Celsius (such as about: 21, 22, 23, 24, 25 degreesCelsius) with a relative atmospheric humidity of from about 45% to about55% (such as about: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%). In some cases, the time period can be from about 1 month to about1 year. In some cases, the time period can be from about 1 month toabout 2 year. In some cases, the time period can be from about 1 monthto about 6 months. In some cases, the time period can be from about 1month to about 3 year. In some cases, the time period can be from about1 month to about 9 months.

The disease or condition can comprise a viral infection such as a SARSvirus infection, a Covid-19 viral infection, or an HCV Genotype 1infection. In some cases, at least a portion of the RNA sequence can beencoded in a coronavirus genome (e.g. a SARS-CoV, a SARS-CoV2, aMERS-CoV, or a CoV-HKU1 genome). The RNA sequence can comprise at leastabout: 70%, 75%, 80%, 85%, 90%, 95% sequence identity to a sequencerecited in SEQ ID NOS: 500-531, 829-831 (Table 6), SEQ ID NOS: 474-499,826-828 (Table 6), SEQ ID NOS: 532-554 (Table 6), or SEQ ID NOS: 555-586(Table 6). In some cases, an RNA sequence can comprise at least about:70%, 75%, 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 476, SEQ IDNO: 481, or SEQ ID NO: 495. The RNA sequence can comprise at leastabout: 5, 10, 15, 20 contiguous bases having at least about: 70%, 75%,80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 476, SEQ ID NO: 481,or SEQ ID NO: 495. In some cases, at least a portion of the RNA sequencecan be encoded in an HCV Genotype 1 genome. The RNA sequence cancomprise at least about: 70%, 75%, 80%, 85%, 90%, 95% sequence identityto SEQ ID NO: 587, SEQ ID NO: 588, or SEQ ID NO: 589. The RNA sequencecan comprise at least about: 5, 10, 15, 20 contiguous bases having atleast about: 70%, 75%, 80%, 85%, 90%, 95% sequence identity to SEQ IDNO: 587, SEQ ID NO: 588, or SEQ ID NO: 589. In some cases, at least aportion of the RNA sequence is encoded in a SARS-CoV-2 virus genome. TheRNA sequence can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%sequence identity to SEQ ID NO: 500, SEQ ID NO: 513, or SEQ ID NO: 518.The RNA sequence can comprise at least about: 5, 10, 15, 20 contiguousbases having at least about: 70%, 75%, 80%, 85%, 90%, 95% sequenceidentity to SEQ ID NO: 500, SEQ ID NO: 513, or SEQ ID NO: 518.

An engineered oligonucleotide or salt thereof can comprise at leastabout: 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to any oneof SEQ ID NOs: 1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45,52-89, 100-154, 184-201, 205-222, 225-233, 235-243, 245-443, 445-453,455-463, 465, 620, 624-825, 835-837, 840-899, and 901-949, or anycombination thereof. An engineered oligonucleotide or salt thereof cancomprise at least about 5, 10, 15, 20 contiguous bases having at leastabout: 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to any oneof SEQ ID NOs: 1-5, 12-14, 19-20, 24-25, 28, 30, 32, 34, 36, 38-45,52-89, 100-154, 184-201, 205-222, 225-233, 235-243, 245-443, 445-453,455-463, 465, 620, 624-825, 835-837, 840-899, and 901-949, or anycombination thereof.

A change in polypeptide expression, such as a fold lower or fold greaterexpression, can be determined by methods as described herein. Forexample, the engineered oligonucleotide or salt thereof can be contactedwith an mRNA sequence and compared to contacting an equivalent amount ofan miR-29 or miR-30 oligonucleotide naturally present in a cell. Thechange in expression can be determined by: (a) transfecting theengineered oligonucleotide into a first isolated cell comprising themRNA sequence, (b) transfecting the miR-29 or miR-30 oligonucleotideinto a second isolated cell comprising the mRNA sequence, and (c)measuring an amount of the polypeptide expressed in the first isolatedcell and the isolated second cell.

A nucleic acid construct can comprise a first strand comprising theengineered oligonucleotide and a second strand comprising a sequencecomplementary to at least a portion of the engineered oligonucleotide.The second strand may be complementary to at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or more of the first strand. The secondstrand may be complementary to at least about: 5, 10, 15, or 20contiguous bases of the first strand. A nucleic acid construct maycomprise an end overhang, such as a 5′ end or a 3′ end. The firststrand, the second strand or a combination thereof may comprise one ormore chemical modifications. At least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% of bases of a first strand, a second strand, or acombination thereof may comprise a chemical modification.

The first strand, the second strand or a combination thereof maycomprise one or more sugar modifications. At least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% of bases of a first strand, a secondstrand, or a combination thereof may comprise a sugar modification. Asugar modification can comprise a glycosylated base. In some cases, abase of a nucleotide can be glycosylated with a glycan. The firststrand, the second strand or a combination thereof may comprise acombination of bases having a chemical modification and a sugarmodification.

In some cases, an engineered passenger oligonucleotide as describedherein can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95% or moresequence identity to any one of SEQ ID NOs: 6-11, 15-18, 21-23, 26-27,29, 31, 33, 35, 37, 46-51, 90-99, 155-183, 202-204, 223-224, 234, 244,444, 454, 464, 466, 838-839, and 900, or any combination thereof. Anengineered passenger oligonucleotide as described herein can comprise atleast about 5, 10, 15, 20 contiguous bases having at least about: 70%,75%, 80%, 85%, 90%, 95% or more sequence identity to any one of SEQ IDNOs: 6-11, 15-18, 21-23, 26-27, 29, 31, 33, 35, 37, 46-51, 90-99,155-183, 202-204, 223-224, 234, 244, 444, 454, 464, 466, 838-839, and900, or any combination thereof.

In some cases, a second strand can comprise a chemically modified baseof a nucleotide. In some cases, a subset of bases of the second strandcan be chemically modified, such as from about 10% to about 5% of bases,from about 1% to about 10% of bases, from about 1% to about 20% ofbases, from about 1% to about 30% of bases, from about 1% to about 40%of bases, from about 1% to about 50% of bases, from about 1% to about60% of bases, from about 1% to about 70% of bases, from about 1% toabout 80% of bases, or from about 1% to about 90% of bases, or more.

A second strand as described herein can be chemically modified in thesame manner as described herein for the engineered oligonucleotide. Insome cases, a second strand, when chemically modified, can have at least90% sequence identity to any one of SEQ ID NOs: 51, 95-99.

A vector can be employed to deliver the engineered oligonucleotide, thenucleic acid construct, or any combination thereof. A vector cancomprise DNA, such as double stranded DNA or single stranded DNA. Avector can comprise RNA. In some cases, the RNA can comprise a basemodification. The vector can comprise a recombinant vector. The vectorcan be a vector that is modified from a naturally occurring vector. Thevector can comprise at least a portion of a non-naturally occurringvector. Any vector can be utilized. In some cases, the vector cancomprise a viral vector, a liposome, a nanoparticle, an exosome, anextracellular vesicle, or any combination thereof. In some cases, aviral vector can comprise an adenoviral vector, an adeno-associatedviral vector (AAV), a lentiviral vector, a retroviral vector, a portionof any of these, or any combination thereof. In some cases, ananoparticle vector can comprise a polymeric-based nanoparticle, anaminolipid based nanoparticle, a metallic nanoparticle (such asgold-based nanoparticle), a portion of any of these, or any combinationthereof. In some cases, a vector can comprise an AAV vector. A vectorcan be modified to include a modified VP1 protein (such as an AAV vectormodified to include a VP1 protein). An AAV can comprise a serotype —suchas an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype,AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, anAAV9 serotype, a derivative of any of these, or any combination thereof.

A pharmaceutical composition can comprise the engineeredoligonucleotide, the nucleic acid construct, the vector, or anycombination thereof. The pharmaceutical composition can further comprisea pharmaceutically acceptable excipient, diluent or carrier. Thepharmaceutical composition can be formulated in a unit dose form. Thepharmaceutical composition can be formulated with a single activeingredient. The pharmaceutical composition can be encapsulated. Thepharmaceutical composition can be formulated as a liquid or as asemi-solid, such as a gel, or a solid. The pharmaceutical compositioncan be formulated at a solution. The pharmaceutical composition can beformulated as an injectable. The pharmaceutical composition can beformulated as a subdermal implant. The pharmaceutical composition can beimplantable. The pharmaceutical composition can be formulated for oraldelivery.

A subject in need thereof may be treated for a disease or condition. Atreatment may be a pre-treatment, a prophylactic treatment, or apreventive treatment. Treatment may include administration to thesubject in need thereof the engineered oligonucleotide, a nucleic acidconstruct, a vector, or a pharmaceutical composition as describedherein. Administration can include delivery via one or more deliveryroutes, such as oral, otic, ocular, rectal or any combination thereof.Administration can include delivery by an intravenous injection, anintramuscular injection, an intrathecal injection, an intraorbitalinjection, a subcutaneous injection, or any combination thereof.Treatment may include more than one engineered oligonucleotide deliveredin a single dose. Delivery can be concurrent delivery, such as deliverymore than one engineered oligonucleotide in a single injection or in twoseparate injections at the same time. Delivery can be sequential, suchas delivery of a first dose and a second dose that can be separated by aperiod of time, such as minutes, hours, days, weeks, or months.

Treatment can include a second therapy or a co-therapy. In cases oftreatment of cancer, a second therapy can include radiation,chemotherapy, an immunotherapy, surgery, or any combination thereof. Incases of treatment of a viral infection, a second therapy can include animmunotherapy, an antiviral, or any combination thereof. In cases oftreatment of fibrosis, a second therapy can include a lifestyle change,an organ transplant, an oxygen therapy, an organ rehabilitation, apharmaceutical composition, or any combination thereof. Administrationof a second therapy can be concurrent or sequential, such as separatedby a period of time such as minutes, hours, days, weeks, or months.

In some cases, a subject may not have been previously diagnosed with adisease or condition. In some cases, a subject may have been diagnosedwith a disease or condition. In some cases, a subject may not havereceived a definitive diagnosis of a disease or condition. In somecases, a subject may have previously had a disease or condition. Asubject may be in remission. A subject may be at risk of developing adisease or condition (such as based at least in part on a previouscondition, a lifestyle factor, a genetic variant, or any combinationthereof). A subject may have received a diagnostic test. A diagnostictest can include an imaging procedure, a blood count analysis, a tissuepathology analysis, a biomarker analysis, or any combination thereof. Insome cases, the disease or condition may be fibrosis or a relatedcondition. In some cases, the disease or condition may be a viralinfection, such as a SARS-CoV infection, a SARS-CoV2 infection, aMERS-CoV infection, a CoV-HKU1 infection, an HIV infection, an HCVinfection, or any combination thereof. In some cases, the disease orcondition may be cancer, such as a head cancer, a neck cancer, skincancer, a cervical cancer, a prostate cancer, or any combinationthereof.

Methods can include in vivo or in vitro delivery methods. Methods caninclude contacting a cell, such as a cell in vivo with the engineeredoligonucleotide, the nucleic acid construct, the vector, or thepharmaceutical composition as described herein. Methods can includecontacting a cell, such as an isolated and purified cell (such as a cellin vitro) with the engineered oligonucleotide, the nucleic acidconstruct, the vector, or the pharmaceutical composition as describedherein. Methods can include contacting a tissue, such as an in vivotissue or an isolated in vitro tissue, with the engineeredoligonucleotide, the nucleic acid construct, the vector, or thepharmaceutical composition as described herein.

A kit can include the engineered oligonucleotide in a container, thenucleic acid construct in a container, the vector in a container, thepharmaceutical composition in a container. A kit can include more thanone engineered oligonucleotide in a container, more than one vector in acontainer, more than one nucleic acid construct in a container, or morethan one pharmaceutical composition in a container. A kit can include aplurality of containers, each container comprising one or moreengineered oligonucleotides, or nucleic acid constructs, or vectors, orpharmaceutical compositions. A kit can include an excipient or a diluentor a buffer or a liquid or gel-like medium for storage of the engineeredoligonucleotide, the nucleic acid construct, the vector, or thepharmaceutical composition. A kit can include an excipient or a diluentor a buffer or a liquid or gel-like medium for in vivo delivery to asubject of the engineered oligonucleotide, the nucleic acid construct,the vector, or the pharmaceutical composition. An excipient or diluentor buffer or liquid or gel-like medium can be included in the containerhousing the engineered oligonucleotide (or nucleic acid construct orvector or pharmaceutical composition) or housed in a separate container.A kit can include a delivery vehicle, such as a syringe or needle. A kitcan include one or more reagents for a downstream analysis.

Referring to FIG. 1A-B, this figure shows natural miR-30 guide strandand passenger strand sequences and examples of engineered familymembers. FIG. 1A shows the sequences of all the natural miR-30 familymembers found in human (i.e. miR-30a-e-5p) which can have tumorsuppressor activity in HNSCC. Sequence variation between the differentfamily members as compared to miR-30a-5p are highlighted in light grey.A guanidine base at position 13 (highlighted in dark gray) represents anon-natural sequence addition comprising a subset of engineered familymembers that can have benefits over natural sequence for drugdevelopment. We have implemented this sequence modification in a subsetof oligonucleotides. FIG. 1B shows the sequence of the mature miR-30a-3ppassenger strand found in human miR-30a duplex. Listed are examples ofnon-natural passenger strand sequence (SEQ ID NO: 6 and 46 through 51)which can be used in combination with the guide strands to create, inpart, the library of miR-30a mimic duplexes. Changes in sequence frommiR-30a-3p are highlighted in grey. The engineered sequencemodifications can be implemented to alter duplex structure andbiochemical characteristics including stability to nucleases andpotency.

Referring to FIG. 2A-C, this figure shows a schematic visualization ofguide strand interactions with cognate target sites in 3′UTRs ofoncogenic mRNAs. FIG. 2A shows free energy (ΔG) and hybridizationbetween natural miR-30a-5p sequence and target sites in the 3′UTRs ofITGA6, SERPINE1, and EGFR transcripts can be predicted using thesoftware RNA hybrid 2.2. FIG. 2B shows free energy (ΔG) andhybridization between engineered family G007-30 (SEQ ID NO: 39)containing the ‘G’ insertion at position 13 and the ITGA6, SERPINE1, andEGFR target sites. Note the lower predicted free energy and greatercomplementarity can infer improved target recognition. FIG. 2C showsthat a mimic containing the same ‘G’ insertion at position 13 as foundin G007-30 can demonstrate improved knockdown of select oncogenictargets. M30-021 and M30-037 duplexes are composed of guide strandsG039-30 (SEQ ID NO: 68) and G011-30 (SEQ ID NO: 55) respectively andcontain the natural miR-30a sequence with certain chemicalmodifications. M30-040 is composed of engineered guide strand G032-30(SEQ ID NO: 65) and M30-048 is composed of guide strand G132-30 (SEQ IDNO: 88), both of which have the G007-30 ‘G’ insertion but with differentchemical modification patterns. UM-SCC-47 cells were plated in 6-wellplates and transfected with 15 nM of the indicated engineered miR-30mimics for 72 hrs. Following treatment, total RNA was collected from thewells and RT-qPCR was performed to determine expression of ITGA6,SERPINE1, and EGFR. HPRT1 served as an endogenous normalization controland expression levels were normalized to negative control (Neg Con)transfections. In each case, M30-040, with the ‘G’ insertion,demonstrated greater knockdown of targeted oncogenes than M30-021 andM30-037 (natural guide strand sequence) and demonstrated as good orgreater knockdown of targeted oncogenes than M30-048, which also has the‘G’ insertion.

Referring to FIG. 3 , this figure shows a schematic visualization ofmiR-30-5p target sites in the MTDH 3′UTR. Shown is an illustration ofthe MTDH mRNA including its coding domain and threeexperimentally-validated miR-30-5p target sites (indicated in grey)within its 3′UTR. Hybridization between natural miR-30a-5p and the MTDHtarget sites, as well as calculated free energy (ΔG) for miR-30-5p andall the indicated engineered guide strands were calculated using thesoftware RNAhybrid 2.2(https://bibiserv.cebitec.uni-bielefeld.de/mahybrid). Net sum of freeenergy across all three target sites (NET) and their averaged ΔG (Avg.)is also indicated. Note all artificial guide strands have lowercalculated net and averaged free energy for hybridization than naturalmiR-30-5p family members inferring improved target recognition.

Referring to Table 8, this table shows calculated free energyrequirements for target recognition of natural and mimic miR-30 familyguide strands to 3′ UTR sites in EGFR, IGF1R, MET and IRS1 genetranscripts. miR-30 mimic exemplary guide strand constructs areindicated include G007-30 (SEQ ID NO: 39) and G064-30 (SEQ ID NO: 43)that have predicted improved base pairing and lower G targeting of theseoncogenes.

TABLE 8 Calculated Free Energy Requirements for Target Recognition ofMimic Guide Strands to miR-30 Downstream Transcript SEQ Free energy(ΔG)* [kcal/mol] ID NO: Guide Strand EGFR IGF1R MET IRS1 1 miR-30a-5p−15.0 −16.4 −10.6 −19.8 2 miR-30b-5p −13.6 −17.0 −11.6 −13.6 3miR-30c-5b −11.8 −17.0 −11.6 −13.6 4 miR-30d-5p −14.8 −14.9 −10.6 −19.85 miR-30e-5p −15.8 −16.4 −11.6 −19.8 628 G079-30 −15.5 −16.5 −10.6 −21.0629 G080-30 −15.2 −15.3 −10.6 −14.0 630 G081-30 −14.2 −15.3 −10.6 −14.0631 G082-30 −13.6 −15.3 −10.6 −13.0 632 G083-30 −12.3 −15.3 −10.6 −13.0633 G084-30 −13.7 −13.9 −10.8 −12.1 634 G085-30 −11.8 −13.9 −10.8 −12.1635 G086-30 −13.6 −13.9 −10.6 −12.1 636 G087-30 −11.8 −13.9 −10.6 −12.139 G007-30 −18.1 −17.4 −10.6 −21.1 624 G075-30 −14.1 −18.0 −14.1 −17.944 G065-30 −13.0 −18.0 −14.1 −17.8 40 G061-30 −16.4 −14.5 −10.6 −19.4 41G062-30 −17.1 −17.4 −12.4 −21.1 42 G063-30 −16.2 −16.4 −11.0 −19.4 637G088-30 −18.5 −16.7 −10.6 −16.1 638 G089-30 −13.0 −17.2 −10.6 −17.3 639G090-30 −17.3 −18.2 −10.6 −18.8 640 G091-30 −13.4 −15.6 −11.1 −21.6 626G077-30 −19.6 −17.2 −10.6 −18.7 627 G078-30 −19.8 −16.7 −10.6 −18.3 641G092-30 −16.1 −17.3 −10.6 −21.4 642 G093-30 −19.3 −17.5 −10.6 −21.0*Free energy (ΔG) was calculated using RNAhybrid 2.2 between theindicated mimic guide strand sequences and target sites in the 3′UTRs ofEGFR (SEQ ID NO: 469), IGF1R (SEQ ID NO: 832), MET (SEQ ID NO: 833), andIRS2 (SEQ ID NO: 834) gene transcripts. Lower predicted free energy (ΔG)infers improved target recognition.

Referring to FIG. 4A-D, this figure shows that engineeredoligonucleotides hybridized to form novel miR-30-5p mimics can haveequivalent or improved anti-tumor activity in cancer cell lines. FIG. 4Ashows UM-SCC-1, UM-SCC-47, and UM-SCC-109 cells were plated in 96-wellplates and were transfected with 15 nM mimics using RNAimax agent 6hours after plating for 5 days. Following treatment, cell viability wasassessed by XTT assay. All data represent the mean±SEM relative to cellstreated in absence of mimic (designated as 100% viability). All mimicscan have unique patterns of chemical modifications and can have one ofthe modified passenger strands. Mimics are separated based on guidesequence including either miR-30a-5p (Native) or miR-G-30-5p derivatives(Engineered). Anti-tumor activity for each mimic can be statistically(P<0.05) an improvement in reducing cell viability or equivalent tonatural miR-30a duplex in each cell line. FIG. 4B shows luciferaseactivity following treatments in a subline of UM-SCC-1 (UM-SCC-1^(luc))genetically engineered to overexpress a luciferase reporter containingcancer-relevant miR-30 target sites within its 3′ untranslated region(UTR). UM-SCC-1^(luc) cells were plated in 96-well plates andtransfected with 15 nM mimics using RNAimax agent 6 hrs after platingfor 3 days before cell lysis and luciferase assay. FIG. 4C displays theanti-tumor activity of native miR-30a and exemplar engineered mimics,M30-043 and M30-046, in additional cell lines with different geneticbackgrounds. Indicated cancer cell lines were plated in 96-well platesand were transfected with 25 nM mimics using RNAimax agent 6 hrs afterplating for 5 days. Following treatment, cell viability was assessed byXTT assay. Statistical significance (P<0.01) compared to Neg Contreatment is indicated. FIG. 4D displays further anti-tumor activity ofa panel of engineered miR-30 mimics in additional cancel cell lines withdifferent histology and genetic backgrounds. Indicated cancer cell lineswere plated in 96-well plates and were transfected with 25 nM mimicsusing RNAimax agent 6 hrs after plating for 5 days. Following treatment,cell viability was assessed by XTT assay. Statistical significance(P<0.01) compared to Neg Con treatment is indicated. All data representthe mean±SEM from a minimum three independent experiments.

Referring to FIG. 5A-B, this figure shows chemical modifications andstructural modifications of engineered mimic duplexes can improve guidestrand and duplex stability to biological nucleases. FIG. 5A shows anexample of Urea-PAGE resolving a nucleic acid marker (M) and samplecomprising mimic duplex, guide strand, and degraded fragments (≤n-1).FIG. 5B shows engineered 10 uM miR-30-5p mimics were incubated in 10%human sera at 37° C. for the indicated lengths of time. Duplex stabilityat each time point was visualized by denaturing Urea-PAGE. Anexceptional example displays that removal of the single nucleotideinternal bulge in M30-040 stabilized the M30-044 mimic duplex tobiological nucleases out to 7 days (168 hrs).

Referring to FIG. 6A-B, this figure shows that modification of mimicsequence and structure can improve stability. FIG. 6A shows engineeredmimics; M30-025, M30-040, and M30-044; which have the identical highlystable engineered guide strand, G032-30 (SEQ ID NO: 65) in duplex withpassenger strands with sequence and chemical modifications which alterthe mimic structure. Indicated is the design schematic towards M30-044incorporating design features that can further enhance mimic stability.Bases in bold contain 2′-O-methyl modifications, lower case bases have2′-flouro substitutions, ‘ps’ signifies phosphothioate backbone and(Amino C6) indicates addition of an amino-carbon 6 chain. FIG. 6B showsmimics were incubated in 10% human sera at 37° C. for the indicatedlengths of time. Duplex stability at each time point was visualized bydenaturing Urea-PAGE. Mimics with enhanced stability can be preferredfor use with conjugation-based delivery systems in which the mimic canbe directly exposed to nucleases in biological fluids and the deliverydoes not provide any additional shielding benefit. M30-040 displayssuperior anti-tumor activity compared to M30-025 or M30-044 aspreviously demonstrated in FIG. 4B.

Referring to FIG. 7A-C, this figure shows the effect of sequence changeson the mimic structure and activity. FIG. 7A displays the sequences ofmimics; M30-033, M30-034, and M30-040. All guide strands have similarchemical modifications (not shown). The same is true for the passengerstrands. M30-033 and M30-034 are comprised of the highly stable guidestrand G042-30 (SEQ ID NO: 69) which contains the natural miR-30a-5pguide sequence. M30-040 is comprised of G032-30 (SEQ ID NO: 65) whichhas identical chemical modifications to G042-30 but has an inserted ‘G’at position 13 (highlighted in grey). M30-033 retains a similarstructure as M30-040 (i.e. internal bulge) and M30-034 has the samepassenger strand as M30-040. FIG. 7B shows a panel of 6 HNSCC cell linesthat were transfected with M30-033, M30-034, M30-040, or a negativecontrol (Neg Con) at 15 nM for 5 days as described above. Cell viabilitywas assessed by XTT assay. Mock treatments were transfected in theabsence of mimic. All data represent the mean±SEM from three independentexperiments. Statistical improvement (P<0.01) in M30-040 reduction incell viability compared to M30-033 (*) and M30-043 (#) were determinedby a two-way t-test. FIG. 7C shows luciferase activity following 15 nMtreatments of UM-SCC-1^(luc) genetically engineered to overexpress aluciferase reporter containing miR-30 target sites within its 3′UTR. Alldata represent the mean±SEM from three independent experiments.Collectively, M30-040 demonstrates a greater reduction in cell viabilityand luciferase knockdown activity.

Referring to FIG. 8A-B, this figure shows anti-tumor activity of miR-30mimic, M30-040, is dependent on guide strand incorporation intoRNA-induced Silencing Complex (RISC). FIG. 8A shows the structure of twomodifications that can be made to the 5′ terminus of the guide strand.In this experiment, two variants of M30-040 were synthesized with eithera 5′-terminal methyl group (5′-OMethyl) or amino-carbon 6 chain(5′-Amino C6) modification of the guide strand, referred to as mimics5′OMe M30-040 and 5′Amino C6 M30-040, respectively. Chemicalmodification of the 5′-hydroxyl terminus in guide strands can attenuateactivity at least in context to siRNA duplexes by blocking intracellularphosphorylation and subsequent recognition by the RISC effector. FIG. 8Bshows UM-SCC-1 and A431 cancer cells treated with each mimic at 15 nMand cell viability was assessed by XTT assay on day 5 as describedabove. Both 5′-terminal modifications interfere with anti-cancer cellactivity of M30-040 in vitro.

Referring to FIG. 9A-C, this figure shows an example development path ofan engineered miR-30 mimic with improved activity. In FIG. 9A, thesequence and structure of mimics M30-037 and M30-043 are composed ofguide strands G011-30 (SEQ ID NO: 55) and G129-30 (SEQ ID NO: 86)respectively and contain the natural miR-30a sequence with the chemicalmodifications as shown. Sequence and structures are also displayed forengineered mimics M30-046 and M30-048 which are composed of guide strandG130-30 (SEQ ID NO: 87) and G132-30 (SEQ ID NO: 88) respectively andcontain an engineered sequence. Its chemical modification pattern wasdesigned to retain poly-targeting activity of downstream transcripts.Indicated is the design schematic towards M30-048 incorporating anaccumulation of features leading to improved activity, including a 3′terminal modification pattern and mimic bulge structure. Bases in boldcontain 2′-O-Methyl modifications, lower case bases have 2′-fluorosubstitutions, ‘ps’ signifies phosphothioate backbone and (Amino C6)indicates addition of an amino-carbon 6 chain. FIG. 9B displaysanti-tumor activity in a panel of cancer cell lines transfected with theindicated mimics or a negative control (Neg Con) at 15 nM for 5 days.Cell viability was assessed by XTT assay. Mock treatments weretransfected in the absence of mimic. All data represent the mean±SEM ofat least three independent experiments. FIG. 9C displays luciferaseactivity following 15 nM treatments of UM-SCC-1^(luc) as describedabove. All data represent the mean±SEM from three independentexperiments.

Referring to FIG. 10A-B, FIG. 10A displays improved anticancer activityand silencing of engineered mimic M30-043 which is composed of guidestrand G129-30 (SEQ ID NO: 86) which is a miR-30 mimic containing thenatural miR-30a sequence with chemical modifications. Compared tounmodified, natural miR-30a, M30-043 has improved activity on cellviability at 7.5 nM in a panel of 7 HNSCC cell lines in vitro.Statistical significance (P<0.05) compared to miR-30a treatment isindicated. FIG. 10B shows luciferase activity following 15 nM treatmentsin a subline of UM-SCC-1 (UM-SCC-1^(luc)) genetically engineered tooverexpress a luciferase reporter containing miR-30 target sites withinits 3′UTR. Mock samples were transfected in the absence of API. All datarepresent the mean±SEM from three independent experiments.

Referring to FIG. 11A-B, this figure displays reductions in innateimmunostimulation for engineered miR-30 mimics. Human Peripheral BloodMononuclear Cells (PBMCs)(˜2-6×10⁵ cells) were plated in round bottom 96well plates and transfected at 133 nM concentrations of the indicatedmimics for 48 hours with RNAiMAX reagent. Levels of TNF-α (FIG. 11A) andIFN-α (FIG. 11B) in supernatant media were quantified by ELISA. Poly(dA:dT) oligonucleotide served as a positive control forimmunostimultation. Transfection with a non-chemically modified RNAduplex (Non-mod) demonstrated potential of mimic immunostimulation.Cells treated in the absence of mimic served to establish baseline.

Referring to FIG. 12A-D, this figure displays sensitization of cancercells to approved treatments, cisplatin and cetuximab, when treated withengineered mimic, M30-040, in combination in vitro. FIG. 12A showsUM-SCC-1 and −46 cancer cells were cultured in the presence ofescalating micro-doses of cisplatin for ≥3 months to create sublinesresistant to cisplatin. Dose response curves evaluating cytotoxicitydemonstrated enhanced resistance to cisplatin compared to parental celllines. FIG. 12B shows parental and cisplatin-resistant cell lines weretransfected with 15 nM M30-040 or nonspecific control mimic (Neg Con)for 5 days as described above. Relative cell number was quantified byXTT. Data is normalized to mock transfection treatments. FIG. 12C showscombination treatment of parental UM-SCC-1 and -46 cells transfectedwith M30-040 at 15 nM for 48 h and then subsequently treated withcisplatin at their respective IC50 concentrations (Cis IC50). Relativecell number was quantified by XTT at day 5 post-transfection. FIG. 12Dshows another cancer cell line, UM-SCC-47, were transfected with 15 nMM30-040 for 48 h and subsequently treated with the indicatedconcentrations of cetuximab. M30-040 pre-treatment may have sensitizedUM-SCC-47 cells to cetuximab.

Referring to FIG. 13A-C, this figure shows anti-tumor activity inorthotopic HNSCC tumors. FIG. 13A shows female SCID mice were implantedwith UM-SCC-1^(Luc) subline in floor-of-mouth on the left side of theiroral cavity. 14 days after tumor implantation, bioluminescence wasmonitored in live animals using In Vivo Imaging System (IVIS) by dosingwith D-luciferin at 150 mg/kg via IP injection. Animals were treated byintravenous injection (IV) with 3 doses BIW at 3 mg/kg with NOV340(PMID: 24832107) LNP-formulated mimics M30-037 and M30-040 or EmptyNOV340 LNP control. Images show tumor bioluminescence from a pair ofanesthetized animals in each treatment group at day 0 prior to firstdose and day 13 of the study. FIG. 13B shows bioluminescence wasquantified for each animal at the indicated time points and meanradiance±SEM was plotted to generate tumor growth curves. Indicated arethe 3 sequential BIW doses of formulated mimic. FIG. 13C female SCIDmice were implanted with UM-SCC-109 tumors on the right flank, grown to˜150 mm3, and subsequently treated by intravenous injection (IV) at 3mg/kg with NOV340 formulated M30-040 or Empty NOV340 LNP control for 6doses on a BIW schedule. Tumor growth curves were plotted by calculatingtumor volume at each time point. A significant growth delay was observedwith M30-040 treatment.

Referring to FIG. 14A-B, this figure shows local tumor treatment withengineered mimic M30-048. FIG. 14A shows Female SCID mice were implantedwith UM-SCC-1^(Luc) subline in floor-of-mouth on the left side of theiroral cavity. 14 days after tumor implantation, bioluminescence wasmonitored in live animals using In Vivo Imaging System (IVIS) by dosingwith D-luciferin at 150 mg/kg via IP injection. Engineered mimic,M30-048, or a Negative control mimic (Neg. Con.) was formulated into anAtelocollagen gel using the AteloGene® local kit (koken). Animals weretreated with a single treatment of 1.5 mg/kg or 3 mg/kg of M30-048 or 3mg/kg of Neg. Con mimic and silencing of luciferase was monitored for 96hrs. FIG. 14B displays quantitation of luciferase signal which iscompletely silenced by 96 hours at both doses.

Referring to FIG. 15 , this figure shows natural miR-29 guide strandsequences and examples of engineered family members. Shown are thesequences of the natural miR-29 family members found in human (i.e.miR-29a-c-3p). Sequence variation between the different family membersas compared to miR-29c-3p are highlighted in grey. Examples ofnon-natural sequences comprising subsets of engineered family memberscan include (i) combining the natural differences between miR-29a-3p andmiR-29b-3p into hybrid sequences (Hybrid); (ii) A-to-G and/or U-to-Gbase mutations at positions 14 and/or 15, respectively (Mutation); (iii)guanidine base insertion at position 15 (Insert.); and (iv) sequencescombining mutation with the base insertion into miR-29b-3p(Mut.+Insert.) or hybrid sequence (Hybrid+Mut.+Insert.). Non-naturalsequence changes are highlighted in light grey. Trimming the 3′ end by1-2 nt is also indicated to comprise additional variants.

Referring to FIG. 16 , this figure shows a schematic visualization of ahuman host cell gene that is an epigenetic modifier and is involvedcancer and reactivation of latent HIV virus .Shown is an illustration ofthe TET1 mRNA including its coding domain and threeexperimentally-validated miR-29 target sites (indicated in grey) withinits 3′UTR. Hybridization between natural miR-29b-3p and the TET1 targetsites, as well as calculated free energy (ΔG) for miR-29-3p and theindicated artificial guide strands were calculated using the softwareRNAhybrid 2.2 (https.//bibiserv.cebitec.uni-bielefeld.de/mahybrid). Netsum of free energy across all three target sites (NET) and theiraveraged ΔG (Avg.) is also indicated. Note all artificial guide strandscan have lower calculated net and averaged free energy for hybridizationthan natural miR-29b-3p inferring improved target recognition. As withthe NEF example depicted in FIG. 22 , the engineered miR-29 guidestrands depicted in FIG. 16 can be used to bind and inhibit an mRNAexpressing a TET1 host protein with a lower predicted free energy (ΔG),a greater specificity, and greater complementarity, than correspondingnatural miRNA sequences lacking the modifications described herein.Since TET1 is important in many disorders included Cancer and viralinfections, this demonstrates that the engineered miR-29 guide strandsmay have improved properties as therapeutic agents by improvingtargeting of TET1.

Referring to FIG. 17 this figure shows miR-29 mimics can have equivalentor greater knockdown activity compared to native miR-29b-1. Luciferaseactivity of a stable subline of HEK cells ( ) genetically engineered tooverexpress a luciferase reporter containing a miR-29 target site withinits 3′UTR. HEK^(luc) cells were plated in 96-well plates and transfectedwith 15 nM mimics using RNAimax agent 6 hrs after plating for 3 daysbefore cell lysis and luciferase assay. Mock treatments were transfectedin the absence of mimic. All data represent mean SEM from threeindependent experiments 72 hours after transfection.

Referring to FIG. 18A-B, this figure shows passenger strand modificationimpacts mimic stability in absence of chemical modifications to theguide strand. FIG. 18A shows sequence comparison of native miR-29b-1duplex to mimics M29-004 and M29-012 in which their passenger strands(top strand) contain three terminal 2′O-methyl nucleotides (bold) onboth ends with an amino-carbon 6 chain (Amino C6) 5′-cap. Duplexcomplementarity was enhanced via sequence alterations in M29-012. Insome cases, chemical modifications can be performed to any of the guidestrands. FIG. 18B shows that reduction in duplex bulges in combinationwith the chemical modification pattern of the passenger strandcontributes to improving duplex stability in absence of additionalchemical modification of the guide strand. 5 μM of miR-29b-1 and themiR-29 mimics were incubated in 10% human sera at 37° C. for theindicated lengths of time. Duplex stability was visualized at theindicated time points by denaturing Urea-PAGE.

Referring to FIG. 19A-B, this figure shows chemical modifications canimprove miR-29 mimic stability. FIG. 19A shows sequence and structure ofexemplar engineered miR-29 mimics with chemical modifications tostabilize them to human nucleases. FIG. 19B shows miR-29 mimics wereincubated in 10% human sera at 37° C. for the indicated lengths of time.Duplex stability at each time point was visualized by denaturingUrea-PAGE. Time points without bands or with prominent smearing areeither missing samples time points or user loading error, respectively.

Referring to FIG. 20A-C, this figure shows passenger strand modificationthat can improve native miR-29a-3p and miR-29b-3p knockdown activity.FIG. 20A shows sequence comparison of native miR-29a duplex with mimicsM29-023 and M29-002, which differ by passenger strand composition. Allthree duplexes contain unmodified, natural miR-29a-3p guide strandsequence. Bases in bold contain 2′-O-methyl. Amino-carbon 6 chain (AminoC6) modification of the 5′ terminus for the indicated passenger strandsis also shown. FIG. 20B shows sequence comparison of native miR-29bduplex with mimics M29-007 and M29-008. All three duplexes contain thesame unmodified, natural miR-29b-3p guide strand sequence. FIG. 20Cshows luciferase activity following treatments of stable subline of HEKcells (HEK^(luc)) genetically engineered to overexpress a luciferasereporter containing a miR-29 target site within its 3′UTR with 15 nMnative miR-29a, native miR-29b, and miR-29 mimics. All data representthe mean±SEM from three independent experiments where cells were assayed72 hours post-transfection. The knockdown activity of mimics containingeither native miR-29a-3p (29a-3p) or miR-29b-3p (29b-3p) can be improvedover natural miR-29a and miR-29b duplexes by hybridizing guide strandswith modified passenger strands.

Referring to FIG. 21 , this figure displays reductions in innateimmunostimulation for engineered miR-29 mimics. Human Peripheral BloodMononuclear Cells (PBMCs)(˜2×10⁵ cells) were plated in round bottom 96well plates and transfected at 133 nM concentrations of the indicatedmimics for 48 hours with RNAiMAX reagent. Levels of IFN-α and TNF-α insupernatant were quantified by ELISA. Poly(dA:dT) oligonucleotide andnon-modified RNA duplex with known strong stimulatory effects (Pos Con)served as positive controls for immunostimulation. Transfection withnon-chemically modified miR-29a and miR-29b duplexes (Non-mod) served ascomparative controls for the indicated example mimics containingchemical modifications. Cells treated in in absence of mimic served toestablish baseline.

Referring to FIG. 22A-B, this figure shows a schematic visualization ofan HIV viral RNA transcript that codes for a viral protein (NEF) and canbe an antiviral target for HIV. FIG. 22A shows free energy (ΔG) andhybridization between natural miR-29b-3p with its cognate target site inthe viral transcript of NEF was calculated using the software RNAhybrid2.2 (https://bibiserv.cebitec.uni-bielefeld.de/mahybrid). For example,artificial miR-29 guide strands consisting of hybrid sequence (G003-29)and/or other non-natural changes (G004-29, G020-29, G027-29, andG031-29) can have lower predicted free energy (ΔG) and greatercomplementarity inferring improved target recognition. Improved targetrecognition, as demonstrated herein, can result in a decrease in a GibbsFree Energy of binding of an engineered oligonucleotide for a targetRNA, can result in an increase specificity of the engineeredoligonucleotide for the target RNA, among a plurality of other RNAsequences, or a combination of these features. Accordingly, theengineered miR-29 guide strands depicted in FIG. 22A can be used to bindand inhibit an mRNA expressing a NEF viral protein with a lowerpredicted free energy (ΔG), a greater specificity, and greatercomplementarity, than corresponding natural miRNA sequences lacking themodifications described herein. Thus, the engineered miR-29 guidestrands can display a potent anti-viral activity against HIV viruses orother virus displaying a NEF viral protein as demonstrated in FIG. 22B.FIG. 22C shows 3*104 CD4+ human t-cells were transfected with 100 nM ofeither control, M29-012 or M29-028 mimic. 24 hours post-transfectioncells were infected with pNL4-HIV, which contains a GFP reporter clonednest to the NEF gene. Relative GFP expression, measured 36 hourspost-infection by flow cytometry, was significantly reduced in cellspre-treated with M29-012 and M29-028.

Referring to FIG. 23A-C, this figure shows knockdown of mRNAs related toHIV replication and latency by miR-29a-3p, miR-29b-3p, M29-002, M29-023,and M29-028. 1×10⁵ Jurkat t-cells were plated in 1 mL of media in 6 wellplates and immediately treated with 100 nM control, natural miR-29a,natural miR-29b, or miR-29 mimics for 72 hours formulated intotransfection agent. Cells were collected and total RNA was purified bydirectzol kits. Following reverse transcription relative expression ofmiR-29 target genes Tet3 (FIG. 23A), Cyclin T1 (FIG. 23B), and DNMT3A(FIG. 23C) was assessed by quantitative RT-PCR. Values represent themean of three biological replicates and error bars represent SEM. *denotes p-value<0.05 by 2 tailed student's T-test. In all cases theengineered mimics of miR-29 have improved knockdown of targeted mRNAthan the natural miRNAs.

Referring to FIG. 24A-B, this figure shows calculated free energyrequirements for target recognition of mimic miR-29 (FIG. 24A) andmiR-30 (FIG. 24B) family guide strands to SARS viral transcript. miR-29mimic exemplary guide strand constructs are indicated. miR-29 guidesequences, for example SEQ. ID. NO. 12 or SEQ. ID. NO. 103, and miR-30guide sequences, for example SEQ. ID. NO. 86 or SEQ. ID. NO. 88, canhave antiviral activity against the SARS virus.

Referring to FIG. 25A-C, this figure shows calculated free energyrequirements for target recognition of mimic miR-29 (FIG. 25A) andmiR-30 (FIG. 25B) family guide strands to SARS-CoV-2 viral transcript.Hybridization and binding free energy (ΔG) between select guide strandsand three predicted targets were calculated using the software RNAhybrid2.2 (https://bibiserv.cebitec.uni-bielefeld.de/mahybrid). Net sum offree energy across all three target sites (NET) and their averaged ΔG(Avg.) is also indicated. FIG. 25C demonstrates the protective effectsof exemplar engineered miR-29 (M29-028) and miR-30 mimics against(M30-043 and M30-48) SARS-CoV-2-induced cell death. 5×10³ Vero cellswere plated in 29 well plates and 6 hrs after plating transfected with25 nM control, M29-028, M30-043 or M30-048 mimics and then infected withSARS-CoV-2 (MOI 0.03) 24 hours later. After another 72 hours, cells wereassayed for viral-induced cytopathic effect using viral toxglo assay(Promega). Pre-treatment with either M29-028, M30-43 or M30-048 resultedin ˜30-40% less viral-induced cell death. miR-29 guide sequences, forexample SEQ. ID. NO. 102 or SEQ. ID. NO. 109, and miR-30 guidesequences, for example SEQ. ID. NO. 86 or SEQ. ID. NO. 88, can haveantiviral activity against the SARS-CoV-2 virus.

Referring to FIG. 26 , this figure shows calculated free energyrequirements for target recognition of mimic miR-29 family guide strandsto HCV Genotype 1 transcript. miR-29 mimic exemplary guide strandconstructs are indicated. miR-29 guide sequences, for example SEQ. ID.NOs. 12, 13, 102, 103, 112, 115, 119, 117, 105, 110, 109, 100, can haveantiviral activity against the HCV Genotype 1.

The antiviral properties of the miR-29, miR-30, or miR-26 families canalso be conserved amongst other RNA-based viruses that contain targetsites within their genome or depend on similar host gene transcripts(e.g. TET1, TET2, TET3, etc.). Based on seed sequence complementarity,26 and 32 candidate target sites in the genomic transcripts of SARS andSARS-CoV-2 coronavirus, respectively. Calculated free energy (ΔG) forthree example targeted sites indicate that many of the artificial familymember can have improved binding potential over native miR-29a-3p,miR-29b-3p, or miR-30a-5p in both SARS (FIG. 24 ) and SARS-CoV-2 (FIG.25 ) genomic transcript. Similarly, artificial family members alsodemonstrated improved calculated binding across three candidate targetsites in Hepatitis C virus (HCV) genome (FIG. 26 ). The mimics,including artificial family members, may have broad acting antiviralproperties at treating or preventing infection of RNA-based viruses.

SEQ ID NOS: 500-531, 829-831 (Table 6) shows miR-29 target sequences ina COVID-19 Coronavirus (SARS-CoV-2) genomic RNA. For example, targetsequences in the COVID-19 genome indicated by SEQ. ID. NO. 500 or SEQ.ID. NO. 502 can be targeted by one or more constructs as describedherein, such as one or more miR-29 constructs.

SEQ ID NOS: 474-499, 826-828 (Table 6) shows miR-29 target sequences ina SARS Coronavirus (SARS-CoV) genomic RNA. For example, target sequencesin the SARS genome indicated by SEQ. ID. NO. 474 or SEQ. ID. NO. 475 canbe targeted by one or more constructs as described herein, such as oneor more miR-29 constructs.

SEQ ID NOS: 532-554 (Table 6) shows miR-29 target sequences in a MERSCoronavirus (MERS-CoV) genomic RNA. For example, target sequences in theMERS genome indicated by SEQ. ID. NO. 535 or SEQ. ID. NO. 537 can betargeted by one or more constructs as described herein, such as one ormore miR-29 constructs.

SEQ ID NOS: 555-586 (Table 6) shows miR-29 target sequences in a HKU1Coronavirus (CoV-HKU1) genomic RNA. For example, target sequences in theHKU1 genome indicated by SEQ. ID. NO. 556 or SEQ. ID. NO. 566 can betargeted by one or more constructs as described herein, such as one ormore miR-29 constructs.

Referring to Table 9 and FIG. 27 , this figure shows engineeredartificial anti-cancer miRNA mimic E1-001 which has no similar naturalncRNA. The Guide strand of E1-001, G001-E1, is engineered to targetsingle and multiple sites in the 3′ UTRs of several oncogenic mRNAs.Table 9 shows calculated free energy requirements for target recognitionof G001-E1 guide strand to the targeted sites in the oncogenic mRNAs.Hybridization and binding free energy (ΔG) were calculated using thesoftware RNAhybrid 2.2(https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). FIG. 27 displaysactivity of engineered artificial miRNA E1-001 in normal non-cancerouscells including fibroblasts, retinal pigment epithelial cell line(RPE-1) or human oral keratinocytes (HOK) cells. Treatments were alsoperformed on various cancer cell lines with different geneticbackgrounds. Indicated cell lines were plated in 96-well plates andtransfected with 7.5 nM E1-001 using RNAimax agent 6 hrs after platingfor 5 days. Following treatment cell viability was assessed by XTTassay. Statistical significance (P<0.01) compared to Neg Con miRNAtreatment is indicated. All Data represent the mean±SEM from a minimumthree independent experiments. Cancer cell lines are significantly moresensitive than non-cancerous cells.

TABLE 9 Calculated binding free energy between ENG- miR-1 guide strandand various targets. SEQ ID NO: Target ΔG 594 CSNK1G1 site 1 −17.4 595CSNK1G1 site 2 −19.1 596 CSNK1G1 site 3 −20.5 597 CSNK1G1 site 4 −21.7598 CSNK1G1 site 5 −21.3 599 CSNK1G1 site 6 −20.1 600 ARHGAP26 site 1−20.3 601 ARHGAP26 site 2 −21.8 602 ARHGAP26 site 3 −17.6 603 ARHGAP26site 4 −20.1 604 ARHGAP26 site 5 −18.1 605 RAB11FIP1 site 1 −27.3 606RAB11FIP1 site 2 −17.2 607 RAB11FIP1 site 3 −17.2 608 RAB11FIP1 site 4−18 608 RBJ site 1 −19.6 609 RBJ site 2 −18 610 SERBP1 site 1 −17.4 611SERBP1 site 2 −18 612 CTBP1 −18.8 613 CRKL −24.7 614 ITGA3 −24.2 615ITGAV −18.4 616 LAMC1 −17.1 617 G6PC2 −19.9 618 PPP2R5E −17.5 619 KLF17−23.9

Referring to Table 10, this table shows engineered miR-205 guide strandsand predicted free energy of binding for cancer-relevant RNAs in the 3′UTRs of ZEB1 (SEQ ID NO: 590), E2F1 (SEQ ID NO: 591), HER3 (SEQ ID NO:592), and SHIP2 (SEQ ID NO: 593) gene transcripts. Lower predicted freeenergy (ΔG) infers improved target recognition. Target HER3 (PMID:19276373) is an oncogene stimulating cancer grown while SHIP2 (PMID:19033458) is a tumor suppressor inhibiting cancer growth. ExemplarEngineered guide strands G016-205 (SEQ ID NO: 260) and G011-205 (SEQ IDNO: 255) and predicted to have stronger targeting of HER3 and weakertargeting of SHIP2.

TABLE 10 Calculated Free Energy Requirements for Target Recognition ofMimic Guide Strands to miR-205 Downstream Transcript SEQ Free energy(ΔG)* [kcal/mol] ID NO: Guide Strand ZEB1 E2F1 HER3 SHIP2 32 miR-205-5p−20.6 −29.3 −28 −30.2 840 G200-205 −22 −24.9 −27.5 −27.5 841 G201-205−19.4 −24.9 −26.8 −29.3 262 G018-205 −19.1 −29.3 −30.4 −30.2 260G016-205 −20.6 −32.7 −31.8 −24.6 247 G003-205 −19.9 −24.9 −32.6 −27.5254 G010-205 −18.4 −24.9 −31.1 −29.3 245 G001-205 −22 −28.3 −31.3 −23.4253 G009-205 19.4 −28.3 −30.6 −23.4 263 G019-205 −19.1 −32.7 −34.4 −27.2248 G004-205 −19.9 −28.3 −36.6 −25.4 255 G011-205 −18.4 −28.3 −34.9−25.4 842 G202-205 −22.6 −25.5 −30.7 −31.4 843 G203-205 −20.1 −25.5−31.8 −31.4 259 G015-205 −20.6 −33.9 −34.7 −27 249 G005-205 −22.4 −30.1−36.7 −27.5 256 G012-205 −20.1 −30.1 −37.8 −27.5 250 G006-205 −22.6−28.9 −34.5 −25.3 257 G013-205 −20.1 −28.9 −35.6 −25.3 246 G002-205−21.1 −23.1 −28 −28.8 844 G204-205 −21.2 −23 −26.5 −28.9 845 G205-205−21.4 −25.5 −33.4 −31.4 261 G017-205 −19.6 −30.9 −34.7 −27.7 251G007-205 −20.4 −30.1 −39.6 −27.5 258 G014-205 −17.6 −30.1 −36.9 −27.5252 G008-205 −21.4 −28.9 −37.4 −25.3 *Free energy (ΔG) was calculatedusing RNAhybrid 2.2 between the indicated mimic guide strand sequencesand target sites in the 3′UTRs of ZEB1 (SEQ ID NO: 590), E2F1 (SEQ IDNO: 591), HER3 (SEQ ID NO: 592), and SHIP2 (SEQ ID NO: 593) genetranscripts. Lower predicted free energy (ΔG) infers improved targetrecognition.

Referring to Table 11, this table shows miR-29 mimic target genes thatmay linked to tissue scarring and fibrosis. Analyses of candidatedownstream target genes within the human genome identified enrichmentfor a number genes that can participate in tissue scarring and fibrosisincluding most members within the collagen superfamily. The miR-29mimics may have further therapeutic potential in fibrotic/scarringpathologies.

TABLE 11 miR-29 Mimic Target Genes Linked to Tissue Scarring andFibrosis Target Genes Gene Ontology Reference (PMID) COL1A1, COL11A1,COL2A1, Collagen Super Family; 25785236; 31231509; COL5A3, COL5A2,COL4A4, Extracellular Matrix 22772564 COL21A1, COL7A1, COL9A1,Remodeling COL19A1, COL5A1, COL22A1, COL8A1, COL4A2, COL6A2, COL24A1,COL4A3, COL4A6, COL25A1, COL16A1, COL15A1 PDGFB, PDGFC, PDGFRBPlatelet-Derived Growth 29155002; 28983598; Factors (PDGF) Signaling27816607; 25678385 WISP1 TGF-b/Wnt Signaling 26867691 LOXL2Collagen/Extracellular Matrix 30986934; 28073888; Remodeling 23821193Elastin Extracellular Matrix 10761639; 30944168 Remodeling TGFB2Transforming growth factor 26704519; 31992593; beta (TGF-b) Signaling31939600; 29615587 HDGF Hepatoma-derived growth 19913322 factor (HDGF)Signaling

Referring to FIG. 28 , this figure shows simultaneous knockdown of DUX4and DBET RNA transcripts in FSHD patient myoblasts by multi-targetedantisense oligonucleotides (ASOs). AS-DX-10 only targets the DUX4transcript, while AS-DX-25, -37, and -55 target both DUX4 and DBETtranscripts. Immortalized 15 Abic myoblast cells were plated in 12-wellplates and the next day transfected with control or targeted ASOs at 50nM using the transfection agent RNAiMAX. One day after platingdifferentiation media was added to induce myoblast formation and DUX4expression. 72 hrs after transfection cells were lysed and total RNA wascollected from the wells and RT-qPCR was performed to determineexpression of DUX4 or DBET transcripts. ASOs AS-DX-25, -37, and -55knockdown both DUX4 and DBET transcripts while AS-DX-10 only knocks downDUX4.

In some cases, as shown in FIG. 30 , a sample 202 containing a geneticmaterial can be obtained from a subject 201, such as a human subject. Asample 202 can be subjected to one or more methods as described herein,such as performing an assay. In some cases, an assay can comprisesequencing (such as nanopore sequencing), genotyping, hybridization,amplification, labeling, or any combination thereof. One or more resultsfrom a method can be input into a processor 204. One or more inputparameters such as a sample identification, subject identification,sample type, a reference, or other information can be input into aprocessor 204. One or more metrics from an assay can be input into aprocessor 204 such that the processor can produce a result, such as adiagnosis of degenerative disc disease or a recommendation for atreatment. A processor can send a result, an input parameter, a metric,a reference, or any combination thereof to a display 205, such as avisual display or graphical user interface. A processor 204 can (i) senda result, an input parameter, a metric, or any combination thereof to aserver 207, (ii) receive a result, an input parameter, a metric, or anycombination thereof from a server 207, (iii) or a combination thereof.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 29 shows acomputer system 101 that is programmed or otherwise configured topredict or confirm efficacy of various constructs for therapeuticeffect, such as in cancer therapeutics or anti-viral therapeutics. Thecomputer system 101 can regulate various aspects of the presentdisclosure, such as, for example, modeling or identifying constructs forvarious therapeutic targets, modeling efficacy or stability ofconstructs, or any combination thereof. The computer system 101 can bean electronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 101 also includes memory or memorylocation 110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 115 (e.g., hard disk), communicationinterface 120 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 125, such as cache, other memory,data storage and/or electronic display adapters. The memory 110, storageunit 115, interface 120 and peripheral devices 125 are in communicationwith the CPU 105 through a communication bus (solid lines), such as amotherboard. The storage unit 115 can be a data storage unit (or datarepository) for storing data. The computer system 101 can be operativelycoupled to a computer network (“network”) 130 with the aid of thecommunication interface 120. The network 130 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 130 in some cases is atelecommunication and/or data network. The network 130 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 130, in some cases with the aid of thecomputer system 101, can implement a peer-to-peer network, which canenable devices coupled to the computer system 101 to behave as a clientor a server.

The CPU 105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions can bestored in a memory location, such as the memory 110. The instructionscan be directed to the CPU 105, which can subsequently program orotherwise configure the CPU 105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 105 can includefetch, decode, execute, and writeback.

The CPU 105 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 115 can store files, such as drivers, libraries andsaved programs. The storage unit 115 can store user data, e.g., userpreferences and user programs. The computer system 101 in some cases caninclude one or more additional data storage units that are external tothe computer system 101, such as located on a remote server that is incommunication with the computer system 101 through an intranet or theInternet.

The computer system 101 can communicate with one or more remote computersystems through the network 130. For instance, the computer system 101can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 101 via the network 130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 101, such as, for example, on the memory110 or electronic storage unit 115. The machine executable ormachine-readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 105. In some cases, thecode can be retrieved from the storage unit 1115 and stored on thememory 110 for ready access by the processor 105. In some situations,the electronic storage unit 115 can be precluded, and machine-executableinstructions are stored on memory 110.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 101, can be embodied in programming. Various aspects of thetechnology can be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which can providenon-transitory storage at any time for the software programming. All orportions of the software can at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, can enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that can bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also can be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, cantake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as can be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediacan take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer can readprogramming code and/or data. Many of these forms of computer readablemedia can be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 101 can include or be in communication with anelectronic display 135 that comprises a user interface (UI) 140 forproviding, for example, one or more results (immediate results orarchived results from a previous method), one or more user inputs, areference value or derivative thereof from a library or database, or anycombination thereof. Examples of UI's include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 105. Thealgorithm can, for example, determine optimized constructs viasupervised learning to optimize therapeutic efficacy, stability, orother attribute of one or more constructs.

While exemplary embodiments have been shown and described herein, suchembodiments are by way of example only. Numerous variations, changes,and substitutions can be performed on the exemplary embodiments. Itshould be understood that various alternatives to the embodimentsdescribed herein may be employed.

What is claimed is:
 1. An engineered oligonucleotide or salt thereofcomprising a polynucleotide sequence from 15-30 nucleotides in length,wherein the engineered oligonucleotide or salt thereof is at leastpartially complementary to at least a portion of at least a first and asecond mRNA originating from two genetic loci that are associated with adisease or condition, wherein the engineered oligonucleotide or saltthereof comprises at least 90% sequence identity to one or moreotherwise comparable mature miR-30 microRNA (miRNA), as determined by aBLAST pairwise sequence alignment algorithm, wherein the engineeredoligonucleotide or salt thereof comprises one or more nucleotideinsertions, nucleotide deletions, nucleotide substitutions, or anycombination thereof, relative to one or more otherwise comparable maturemiR-30 miRNA, wherein the engineered oligonucleotide or salt thereofcomprises at least about 10% lower Gibbs free energy (ΔG) of binding atabout 37 degrees Celsius and at a pH ranging from about 7.2 to about7.6, relative to a ΔG of binding of the otherwise comparable maturemiR-30 miRNA binding to the first and the second mRNA at about 37degrees Celsius and at a pH ranging from about 7.2 to about 7.6.
 2. Theengineered oligonucleotide or salt thereof of claim 1, wherein theengineered oligonucleotide or salt thereof has at least 95% sequenceidentity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 624, SEQ ID NO: 625, SEQ ID NO: 626, SEQ ID NO: 627, SEQ IDNO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632,SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ IDNO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641,SEQ ID NO: 642, or SEQ ID NO: 643, as determined by a BLAST pairwisesequence alignment algorithm.
 3. The engineered oligonucleotide or saltthereof of claim 1, wherein the engineered oligonucleotide or saltthereof independently comprises a chemically modified base, chemicallymodified sugar, chemically modified backbone or phosphate linkage, orany combination thereof relative to a naturally occurring base, sugar,backbone, or phosphate linkage, wherein the chemical modification isselected from the group consisting of: a ribose sugar, a deoxyribosesugar, a methyl group, a fluoro group, a methoxyethyl group, an ethylgroup, a hydroxymethyl group, a formyl group, bridged nucleic acid,locked nucleic acid, a carboxylic acid or salt thereof, aphosphothionate modified backbone, a methylphosphonate modifiedbackbone, an amino-alkyl chain modification, and any combinationthereof.
 4. The engineered oligonucleotide or salt thereof of claim 1,wherein the engineered oligonucleotide sequence further comprises acovalent linker conjugated to an antibody, a naturally occurring ligand,a small molecule, or a peptide.
 5. The engineered oligonucleotide orsalt thereof of claim 1, wherein the engineered oligonucleotide or saltthereof when contacted with the first or the second mRNA sequenceproduces from about 1.2-fold to about 10-fold lower expression of thepolypeptide encoded by the first or the second mRNA sequence, ascompared to contacting the equivalent amount of the miR-30 miRNA; asdetermined by: a) transfecting the engineered oligonucleotide or saltthereof into the first isolated mammalian cell comprising the first orthe second mRNA sequence, b) transfecting the miR-30 miRNA into thesecond isolated mammalian cell comprising the first or the second mRNAsequence, and c) measuring the amount of the polypeptide expressed inthe first isolated mammalian cell and the second isolated mammaliancell.
 6. The engineered oligonucleotide or salt thereof of claim 1,wherein the first mRNA is an RNA encoded by ABL1, ABL2, AFAP1L2, AKT3,ARHGAP26, ATF1, BCL2, BCL6, CBLB, CCNA1, CD80, COL25A1, CRKL, CSNK1G,CTHRC1, DEK, EGFR, FAP, GJA1, GLDC, IGF1R, ILlA, IRF4, IRS1, ITGA6,KRAS, LMO2, LOX, MAF, MET, MTDH, MYBL2, MYCN, NT5E, PDGFB, PDGFRB, PIM1,PLAGI, PPP2R5E, SERPINE1, SOCS1, STAT1, THBS2, or WNT5A.
 7. Theengineered oligonucleotide or salt thereof of claim 1, wherein thesecond mRNA is an RNA encoded by ABL1, ABL2, AFAP1L2, AKT3, ARHGAP26,ATF1, BCL2, BCL6, CBLB, CCNA1, CD80, COL25A1, CRKL, CSNK1G, CTHRC1, DEK,EGFR, FAP, GJA1, GLDC, IGF1R, ILlA, IRF4, IRS1, ITGA6, KRAS, LMO2, LOX,MAF, MET, MTDH, MYBL2, MYCN, NT5E, PDGFB, PDGFRB, PIM1, PLAGI, PPP2R5E,SERPINE1, SOCS1, STAT1, THBS2, or WNT5A.
 8. An engineered passengeroligonucleotide or salt thereof comprising a polynucleotide sequencefrom 15-30 nucleotides in length, wherein the engineered passengeroligonucleotide or salt thereof is at least partially complementary toat least a portion of the engineered oligonucleotide or salt thereofclaim
 1. 9. The engineered passenger oligonucleotide or salt thereof ofclaim 8, wherein the engineered passenger oligonucleotide or saltthereof independently comprises a chemically modified base, chemicallymodified sugar, chemically modified backbone or phosphate linkage, orany combination thereof relative to a naturally occurring base, sugar,backbone, or phosphate linkage, wherein the chemical modification isselected from the group consisting of: a ribose sugar, a deoxyribosesugar, a methyl group, a fluoro group, a methoxyethyl group, an ethylgroup, a hydroxymethyl group, a formyl group, a carboxylic acid or saltthereof, a phosphothionate modified backbone, a methylphosphonatemodified backbone, an amino-alkyl chain modification, and anycombination thereof.
 10. The engineered passenger oligonucleotide orsalt thereof of claim 8, wherein the engineered passengeroligonucleotide or salt thereof has at least 95% sequence identity toany one of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, or SEQ ID NO: 51, SEQ ID NO: 90, SEQ ID NO: 91, SEQ IDNO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 900, asdetermined by a BLAST pairwise sequence alignment algorithm.
 11. Theengineered passenger oligonucleotide or salt thereof of claim 8, whereinthe engineered passenger oligonucleotide or salt thereof furthercomprises a covalent linker conjugated to an antibody, a naturallyoccurring ligand, a small molecule, or a peptide.
 12. The engineeredpassenger oligonucleotide or salt thereof of claim 9, wherein thestructure and chemistry is optimized to impart greater than or equal to100X stability to natural nucleases compared to an unmodified sequenceor a comparable miR-30 miRNA.
 13. A nucleic acid construct comprising:(a) a first strand comprising an engineered oligonucleotide or saltthereof that has at least 95% sequence identity to any one of SEQ ID NO:38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 624, SEQ ID NO: 625,SEQ ID NO: 626, SEQ ID NO: 627, SEQ ID NO: 628, SEQ ID NO: 629, SEQ IDNO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634,SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ IDNO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, or SEQ ID NO:643, and (b) a second strand comprising an engineered passengeroligonucleotide or salt thereof that has at least 95% sequence identityto any one of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:49, SEQ ID NO: 50, or SEQ ID NO: 51, SEQ ID NO: 90, SEQ ID NO: 91, SEQID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 900 with asequence complementary to at least a portion of the first strand,wherein the first strand and the second strand are independently from15-30 nucleotides in length.
 14. A vector that comprises the nucleicacid construct of claim
 13. 15. The vector of claim 14, wherein thevector is present in a liposome, a nanoparticle, or both.
 16. A vectorthat comprises the engineered oligonucleotide or salt thereof of claim14, wherein the vector is a viral vector, and optionally wherein theviral vector is an adeno-associated viral (AAV) vector.
 17. An isolatedcell that comprises the engineered oligonucleotide of claim
 1. 18. Apharmaceutical composition in unit dose form comprising the engineeredoligonucleotide of claim 1 and a pharmaceutically acceptable: excipient,diluent, or carrier.
 19. A method of treating a disease or condition ina subject in need thereof the method comprising: administering to thesubject in need thereof a therapeutically effective amount of thepharmaceutical composition of claim 18, thereby treating the disease orcondition.
 20. The method of claim 19, wherein the disease or conditionis a cancer, a head cancer, a neck cancer, a skin cancer, a cervicalcancer, a prostate cancer, a fibrosis, a viral infection, a coronavirus,a SARS-CoV infection, a SARS-COV-2 infection, a MERS-CoV infection, aCoV-HKU1 infection, an HIV infection, or an HCV infection.
 21. Themethod of claim 19, wherein the subject in need thereof has beendiagnosed with a disease or condition by a diagnostic test.
 22. A kitthat comprises the pharmaceutical composition of claim 18 in acontainer.